Methods, compositions, kits, and uses for analysis of nucleic acids comprising repeating a/t-rich segments

ABSTRACT

Described herein are methods, compositions, kits, and uses thereof for analysis of nucleic acid segments comprising a repeating A/T-rich segment, wherein the repeating A/T-rich segment is: (i) a homopolymeric segment comprising at least 10 A residues, at least 10 T residues, or at least 10 U residues, wherein the at least 10 A, T, or U residues are consecutive or interrupted once by one to three other nucleotides; or (ii) a segment comprising (T n A) m , (AT n ) m , (TA n ) m , or (A n T) m , wherein n is 2 or greater and m is such that the length of the repeating A/T-rich segment is 10 or more residues.

This is a divisional of application Ser. No. 15/744,149, filed Sep. 26,2018, which is a national stage application under 3 U.S.C. § 371 ofInternational Application No. PCT/US2016/043503, filed Jul. 22, 2016,which claims the benefit of U.S. Provisional Application No. 62/196,239,filed Jul. 23, 2015, each of which is incorporated herein by reference.

The present application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 26, 2016, isnamed 10256_0050-00304_SL.txt and is 1,867 bytes in size.

This invention is in the field of nucleic acid analysis. In particular,the invention relates to improved methods for analyzing nucleic acidscomprising repeating A/T-rich segments.

Many molecular biology techniques involve nucleic acid synthesis, e.g.,synthesis of DNA or RNA. Nucleic acid synthesis therefore plays acentral role in numerous biotechnological, medical, and researchdiscovery applications. For example, polymerase chain reaction (PCR) isa DNA synthesis reaction that rapidly amplifies DNA template molecules.A typical PCR reaction mixture comprises primer sequences which arecomplementary to the ends of a desired template, deoxynucleotidetriphosphates (dNTPs), various buffer components, and a DNA polymerase.The reaction mixture is admixed with a DNA sample known or suspected ofharboring the desired template. The resulting mixture is then subjectedthrough repeated cycles of template denaturation, primer annealing tothe denatured template, and primer extension by the DNA polymerase,creating copies of the template. Because the product of each cycle canact as a template for subsequent reaction cycles, amplificationgenerally proceeds in an exponential fashion. See, e.g., U.S. Pat. No.4,683,202, and M. J. McPherson & S. G. Moller, PCR: The Basics (2nd Ed.,Taylor & Francisco) (2006). PCR is a widely used technique due to itsrapidity, low cost, sensitivity, and adaptability to high-throughputapplications and automation.

A notable application of PCR is the detection and analysis of repetitivenucleotide sequences which can occur in the genome. Analysis ofrepeating A/T-rich segments, including homopolymeric repeat sequences ofhighly variable lengths (e.g., repeat length polymorphisms), can beuseful for, e.g., genotyping, forensics, diagnostics, populationgenetics, and taxonomic studies.

For example, certain repeat length polymorphisms are known to beassociated with disease states. Detection of such polymorphisms cantherefore be helpful in disease diagnosis and treatment. For example,intron 6 of the TOMM40 gene contains a poly-T repeat length polymorphism(rs10524523), which has potential applications in Alzheimer's disease(AD) diagnosis. TOMM40 is also known as TOM40, PEREC-1, PER-EC1,C19orf1, D19S1177E, or P38.5. Three allelic categories were defined forthis locus based on variation in its poly-T repeat length: Short (S,T≤19), Long (L, 20≤T≤29) and ‘Very Long’ (VL, T≥30). See Roses et. al.,Alzheimer's & Dementia 9:132-136 (2013). The TOMM40 poly-T sizepolymorphism was recently reported as being associated with late-onsetAlzheimer's disease (LOAD) and with cognitive performance in theelderly. See Roses et. al., The Pharmacogenetics Journal 10:375-384(2010); Alzheimer's & Dementia 9:132-136 (2013).

An unfortunate limitation to the accuracy of DNA synthesis is theproblem of polymerase slippage and stuttering. Repeating A/T-richsegments, such as homopolymeric nucleic acid segments (also referred toas mononucleotide repeat regions), are particularly susceptible toslippage and stuttering events. During polymerase slippage, thepolymerase stalls and dissociates from the template strand duringreplication of the repeating A/T-rich segment, resulting in separationof the growing strand from the template strand. Slippage often thengives rise to polymerase stuttering, wherein the growing strandre-anneals to the template strand in an out-of-register manner such thatone or more bases in either the growing or template strand are unpaired,forming a bubble. Such bubble formation at the growing or templatestrand results in expansion or truncation of the repeating A/T-richsegment, respectively (sometimes referred to as frameshift error). Inparticular, bubble formation on the growing or primer strand results inexpansion of the repeating NT-rich segment. And bubble formation on thetemplate strand results in contraction of the repeating A/T-richsegment. Polymerase slippage and stuttering are known to cause higherror rates in amplification and analysis of repeating A/T-richsegments. For example, slippage during PCR amplification can generate acomplex mixture of amplicons of varying lengths, making it difficult toaccurately determine the length of the repeating A/T-rich segment. Asnoted by Fazekas et. al. with respect to homopolymeric segments, “as therepeat number increases, the number of ambiguous bases increasesdisproportionately . . . to the point where sequence data cannot be usedat all past the repeat.” See Fazekas et. al., Taxon 59(3):694-697 at 694(2010).

The difficulty due to slippage and stuttering may be compounded in thecase of samples containing two alleles with repeating A/T-rich segmentsof similar lengths, e.g., lengths differing by a number of nucleotidessuch as 1, 2, 3, or 4. In such cases, slippage and stuttering may makeit difficult to distinguish the products of the longer and shorteralleles, such that determining whether a sample is, e.g., homozygous foran allele with a repeating A/T-rich segment of length n or heterozygousfor alleles with repeating A/T-rich segments of length n−1 and n+1 maynot be possible, or may not be possible with high confidence.

Attempts have been made to mitigate the problem of slippage/stuttering.See Fazekas et al., BioTechniques 48:277-285 (2010). There, improvedsequence quality was reported only for homopolymeric segments that are15 nucleotides or less. “In . . . samples that possessed repeats greaterthan 15 bp, the sequence quality was not improved.” See BioTechniques48:277-285 at 695 (2010). Other attempts to improve amplification ofhomopolymeric segments by including a portion of the repeat region inthe primer sequence were reported to “improve scoring of . . . repeatsless than 20 bp.” See Flores-Renteria et al., American Journal ofBotany: e1-e3 (2011) at e2 (doi:10.3732/ajb.1000428).

Due to the issues of polymerase slippage/stuttering, TOMM40 poly-Tpolymorphisms are difficult to amplify and genotype, particularly thosein the L (20≤T≤29) and VL (T≥30) categories. Because of this difficulty,researchers have been forced to engage in data manipulation in order toclassify the polymorphic alleles. Such data manipulation includes, e.g.,(1) calling only the most abundant peak in a complex distribution ofamplicon sizes, or (2) using pattern recognition algorithms to matchpeak patterns to known genotypes (e.g., genotypes obtained from knownclonal populations). Therefore, there exists a need for methods thatreduce slippage/stutter, including methods that reduce slippage/stutterin synthesis of repeating A/T-rich segments such as homopolymericsegments, including long repeating A/T-rich segments or homopolymericsegments (e.g., greater than about 15 nucleotides, or about 20nucleotides or greater). Such methods can be used for accurate analysisof nucleic acids containing repeating A/T-rich segments such ashomopolymeric segments. Such methods can also be used for analysis ofrepeat length polymorphisms such as, e.g., the TOMM40 poly-Tpolymorphism.

In an embodiment, provided is a method of extending at least one nucleicacid template comprising a repeating A/T-rich segment, the methodcomprising performing a nucleic acid amplification reaction in anaqueous solution comprising the at least one nucleic acid template; atleast one polymerase; at least one primer; magnesium; and NTPs in anAT/GC ratio of about 2 or higher; wherein the repeating A/T-rich segmentis: (i) a homopolymeric segment comprising at least 10 A residues, atleast 10 T residues, or at least 10 U residues, wherein the at least 10A, T, or U residues are consecutive or interrupted once by one to threeother nucleotides; or (ii) a segment comprising (T_(n)A)_(m),(AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m), wherein n is 2 or greaterand m is such that the length of the repeating A/T-rich segment is 10 ormore residues.

In an embodiment, provided is a method of amplifying at least one DNAtemplate comprising a homopolymeric segment, the method comprisingperforming a DNA amplification reaction in an aqueous solutioncomprising the at least one DNA template; at least one hot-start DNApolymerase; at least two primers; magnesium at a concentration in therange from 1.5 mM to 3 mM; dNTPs in an AT/GC ratio of 5 or higher and atotal concentration in the range from 1500 μM to 2500 μM; wherein thehomopolymeric segment comprises at least 12 consecutive A residues or atleast 12 consecutive T residues.

In an embodiment, provided is a method of detecting a genotypeassociated with late-onset Alzheimer's disease, comprising performing aDNA amplification reaction on at least one genetic locus associated withlate-onset Alzheimer's disease, the genetic locus comprising ahomopolymeric segment of at least 10 consecutive A residues or at least10 consecutive T residues, wherein the DNA amplification reaction isperformed in an aqueous solution comprising at least one DNA polymerase;at least two primers; magnesium; and dNTPs in an AT/GC ratio of 2 orhigher; and wherein the DNA amplification reaction produces a productcomprising a homopolymeric segment of at least 10 consecutive A residuesor at least 10 consecutive T residues.

In an embodiment, provided is a kit comprising at least two distinctprimers and NTPs in an AT/GC ratio greater than 2, the at least twoprimers being suitable for amplifying a genetic locus comprising eitherof (i) a homopolymeric segment of at least 10 consecutive A residues orat least 10 consecutive T residues or (ii) a repeating A/T-rich segmentcomprising (T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m),wherein n is 2 or greater and m is such that the length of the repeatingA/T-rich segment is 10 or more residues.

In an embodiment, provided is a kit comprising reagents for use inamplifying at least one template comprising either of (i) ahomopolymeric segment of at least 10 consecutive A residues or at least10 consecutive T residues or (ii) a repeating A/T-rich segmentcomprising (T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m),wherein n is 2 or greater and m is such that the length of the repeatingA/T-rich segment is 10 or more residues, wherein the reagents compriseNTPs in an AT/GC ratio greater than 2.

In an embodiment, provided is a reaction solution comprising at leastone polymerase; one or more primers; magnesium; and NTPs in an AT/GCratio of 2 or higher.

In an embodiment, provided is a use of NTPs in an AT/GC ratio of 2 orhigher for amplifying a nucleic acid template comprising a repeatingA/T-rich segment that is: (i) a homopolymeric segment of at least 10 Aresidues, at least 10 T residues, or at least 10 U residues, wherein theat least 10 A, T, or U residues are consecutive or interrupted once byone to three other nucleotides; or (ii) a segment comprising(T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m), wherein n is2 or greater and m is such that the length of the repeating A/T-richsegment is 10 or more residues.

BRIEF DESCRIPTION OF DRAWING(S)

FIG. 1A depicts an exemplary workflow of a method disclosed herein.

FIG. 1B depicts an exemplary reaction solution disclosed herein.

FIG. 2 depicts an exemplary desired capillary electrophoresis (CE) peakprofile and an exemplary undesired CE peak profile for two alleles withsimilar repeat numbers.

FIG. 3A depicts capillary electrophoresis results from an experimenttesting the effect of AT/GC biased ratios on polymeraseslippage/stutter, using DNA standard samples containing 35 T/36 Talleles for the TOMM40 poly-T polymorphism. Information regardingexperimental conditions for the data in this and subsequent figures isprovided in the Examples section below.

FIG. 3B depicts capillary electrophoresis results from an experimenttesting the effect of AT/GC biased ratios on polymeraseslippage/stutter, using DNA standard samples containing 16 T/36 Talleles for the TOMM40 poly-T polymorphism, in the vicinity of the 16 Tpeak.

FIG. 3C depicts capillary electrophoresis results from an experimenttesting the effect of AT/GC biased ratios on polymeraseslippage/stutter, using DNA standard samples containing 16 T/36 Talleles for the TOMM40 poly-T polymorphism, in the vicinity of the 36 Tpeak.

FIG. 3D depicts capillary electrophoresis results from an experimenttesting the effect of AT/GC biased ratios on polymeraseslippage/stutter, using DNA standard samples containing 34 T/36 Talleles for the TOMM40 poly-T polymorphism.

FIG. 4 depicts capillary electrophoresis results from an experimenttesting the effects of Mg²⁺ concentration and total dNTP concentrationon polymerase slippage/stutter.

FIG. 5 depicts capillary electrophoresis results from an experimenttesting the effects of Mg²⁺ concentration, total dNTP concentration, andAT/GC biased ratios on polymerase slippage/stutter.

FIG. 6 depicts the effects of DMSO and betaine titration on polymeraseslippage/stutter during amplification of a 16 T TOMM40 polymorphism.

FIG. 7 depicts the effects of DMSO and betaine titration on polymeraseslippage/stutter during amplification of a 36 T TOMM40 polymorphism.

FIG. 8 depicts the effects of reduced PCR cycles on polymeraseslippage/stutter during amplification of a 16 T/36 T TOMM40polymorphism.

FIG. 9 depicts the effects of reduced PCR cycles on polymeraseslippage/stutter during amplification of a 34 T/36 T TOMM40polymorphism.

FIG. 10 depicts the effects of an increased AT/GC concentration ratio,lowered PCR cycle number (all in Condition B, relative to Condition A),1M Betaine, and 1% DMSO on detection of short (16 T) and very long (36T) TOMM40 polymorphic alleles.

FIG. 11 depicts the effects of an increased AT/GC concentration ratio,lowered PCR cycle number, 1M Betaine, and 1% DMSO (all in Condition B,relative to Condition A) on detection of long polymorphic poly-T allelesthat have adjacent lengths.

FIG. 12 depicts the effects of an increased AT/GC concentration ratio,lowered PCR cycle number, 1M Betaine, and 1% DMSO (all in Condition B,relative to Condition A) on detection of long polymorphic poly-T allelesthat are separated in length by 1 nucleotide.

FIG. 13A shows products amplified from RS1310 (35 T/36 T) samples usingcondition C.

FIG. 13B shows products amplified from RS1310 (35 T/36 T) samples usingcondition B.

FIG. 14A shows products amplified from RS1311 (16 T/36 T) samples usingcondition C.

FIG. 14B shows products amplified from RS1311 (16 T/36 T) samples usingcondition B.

FIG. 15A shows products amplified from RS1317 (29 T/36 T) samples usingcondition C.

FIG. 15B shows products amplified from RS1317 (29 T/36 T) samples usingcondition B.

FIG. 16A shows products amplified from RS1318 (16 T) samples usingcondition C.

FIG. 16B shows products amplified from RS1318 (16 T) samples usingcondition B.

FIG. 17A shows products amplified from RS1319 (34 T/36 T) samples usingcondition C.

FIG. 17B shows products amplified from RS1319 (34 T/36 T) samples usingcondition B.

FIG. 18A shows products amplified from NA07541 (34 T/38 T) samples usingcondition C.

FIG. 18B shows products amplified from NA07541 (34 T/38 T) samples usingcondition B.

FIG. 19A shows products amplified from NA20243 (16 T/20 T) samples usingcondition C.

FIG. 19B shows products amplified from NA20243 (16 T/20 T) samples usingcondition B.

FIG. 20A shows products from a synthetic DNA template containing a 48 Thomopolymeric segment amplified using condition A.

FIG. 20B shows products from a synthetic DNA template containing a 48 Thomopolymeric segment amplified using condition B.

DETAILED DESCRIPTION

The use of the word “a”, “an” or “the” when used in conjunction with theterm “comprising” in the claims and/or the specification can mean “one,”but it is also consistent with the meaning of “one or more,” “at leastone,” and “one or more than one.” In this application, the use of thesingular includes the plural unless specifically stated otherwise. Alsoin this application, the use of “or” means “and/or” unless statedotherwise. Furthermore, the use of the term “including,” as well asother forms, such as “includes” and “included,” are not limiting. Anyrange described herein will be understood to include the endpoints andall values between the endpoints.

The term “nucleotides” refers to molecules or ions capable of formingnucleic acids. Nucleotides can comprise a base moiety, a sugar moiety,and one or more phosphates (e.g., diphosphate or triphosphate). Thesugar moiety can be deoxyribose, ribose, or another sugar moiety. Thesugar moiety can be a modified sugar moiety, e.g., wherein one or moreof the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the like. Exemplarybase moieties include purine and pyrimidine bases, and otherheterocyclic bases that have been modified. Exemplary modified basesinclude, e.g., methylated purines, methylated pyrimidines, acylatedpurines or pyrimidines, alkylated riboses, and other heterocycles.Nucleotides can also comprise labeled moieties, such as those labeledwith hapten, biotin, fluorescent, or chemiluminescent labels.

“NTP” refers to any nucleotide triphosphate, including ribonucleotidetriphosphates (rNTPs) and deoxyribonucleotide triphosphates (dNTPs) andanalogs of a nucleotide triphosphate. Deoxyribonucleotide triphosphatesinclude, e.g., dATP, dCTP, dGTP, dTTP, dUTP, and analogs thereof. Asused herein, a “dNTP mix” refers to a mix of two or more of dATP, dCTP,dCTP, dTTP, dUTP, and analogs thereof. Similarly, ribonucleotidetriphosphates include, e.g., rATP, rCTP, rGTP, rTTP, rUTP, and analogsthereof. As used herein, an “rNTP mix” refers to a mix of two or more ofrATP, rCTP, rCTP, rTTP, rUTP, and analogs thereof.

The term “AT/GC ratio” refers to the ratio of (i) the sum of theconcentrations of ATP, TTP, UTP, and any analogs thereof, to (ii) thesum of the concentrations of CTP, GTP, and any analogs thereof, in agiven solution or mixture. As noted above, an “NTP” includes rNTPs anddNTPs. Thus, for example, ATP includes rATP and dATP.

“Nucleotide analogs” refer to molecules or ions comprising a base moietyother than the natural bases adenine (A), cytosine (C), guanine (G),thymine (T), or uracil (U), a sugar moiety (which can be identical orsimilar to deoxyribose or ribose), and at least one phosphate ormultiple phosphate (e.g., diphosphate or triphosphate) moiety. Thenucleotide analog can be an analog of a specific nucleotide, such asATP, CTP, GTP, TTP, or UTP, when it comprises a triphosphate and a sugarmoiety, the structure and configuration of both of which are suitablefor incorporation into a nucleic acid double helix by a polymerase, anda base whose base pairing properties in a nucleic acid double helix andloci of incorporation by DNA polymerases in a nucleic acid double helixare most similar to one of the five previously listed nucleotides, withthe exception that analogs of TTP can also be analogs of UTP and viceversa.

The terms “template”, “template strand”, and “template nucleic acid” areused interchangeably herein to refer to a nucleic acid that is bound bya primer for extension by a nucleic acid synthesis reaction.

The term “locus” refers to a gene, nucleotide, or sequence on achromosome. A locus can be “polymorphic” or exhibit a “polymorphism” ifalternative forms of the locus exist in a population. An “allele” of alocus, as used herein, refers to a species of the locus.

The term “repeating A/T-rich segment” as used herein refers to ahomopolymeric segment, defined below, or a segment comprising(T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m), wherein n is2 or greater and m is such that the length of the repeating A/T-richsegment is 10 or more residues. The value of n need not be constantthroughout the segment. Thus, examples of repeating A/T-rich segmentsinclude AATAATAATAAT (SEQ ID NO: 3), AATAAATAAT (SEQ ID NO: 4),AAATAAAAAT (SEQ ID NO: 5), AATAAAAAAT (SEQ ID NO: 6), etc. With respectto a segment comprising (T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or(A_(n)T)_(m), in some embodiments, n is a value ranging from 2 to 10. Insome embodiments, n is a value ranging from 3 to 10. In someembodiments, n is a value ranging from 4 to 10. In some embodiments, nis a value ranging from 2 to 8. In some embodiments, n is a valueranging from 3 to 8. In some embodiments, n is a value ranging from 4 to8. In some embodiments, n is a value ranging from 2 to 6. In someembodiments, n is a value ranging from 3 to 6. In some embodiments, m isa value ranging from 2 to 20. In some embodiments, m is a value rangingfrom 3 to 20. In some embodiments, m is a value ranging from 4 to 20. Insome embodiments, m is a value ranging from 2 to 15. In someembodiments, m is a value ranging from 3 to 15. In some embodiments, mis a value ranging from 4 to 15. In some embodiments, m is a valueranging from 2 to 10. In some embodiments, m is a value ranging from 3to 10. In some embodiments, m is a value ranging from 4 to 10. In someembodiments, m is a value ranging from 2 to 8. In some embodiments, m isa value ranging from 3 to 8. In some embodiments, m is a value rangingfrom 4 to 8. In some embodiments, the length of the repeating A/T-richsegment is in the range from about 10 to about 60 residues. In someembodiments, the length of the repeating A/T-rich segment is in therange from about 10 to about 40 consecutive residues. In someembodiments, the length of the repeating A/T-rich segment is in therange from about 15 to about 40 consecutive residues. In someembodiments, the length of the repeating A/T-rich segment is in therange from about 20 to about 40 consecutive residues. In someembodiments, the length of the repeating A/T-rich segment is in therange from about 5 to about 50 consecutive residues. In someembodiments, the length of the repeating NT-rich segment is in the rangefrom about 10 to about 50 consecutive residues. In some embodiments, thelength of the repeating A/T-rich segment is in the range from about 15to about 50 consecutive residues. In some embodiments, the length of therepeating A/T-rich segment is in the range from about 20 to about 50consecutive residues. In some embodiments, the length of the repeatingA/T-rich segment is in the range from about 5 to about 60 consecutiveresidues. In some embodiments, the length of the repeating A/T-richsegment is in the range from about 10 to about 60 consecutive residues.In some embodiments, the length of the repeating A/T-rich segment is inthe range from about 15 to about 60 consecutive residues. In someembodiments, the length of the repeating A/T-rich segment is in therange from about 20 to about 60 consecutive residues. Unless otherwiseindicated, a repeating A/T-rich segment can comprise an interruption asexplained in the following paragraph. In some embodiments, a repeatingA/T-rich segment does not comprise an interruption.

The term “homopolymeric segment” as used herein refers to segments ofnucleic acid which comprise a nucleotide such as A, T, or U repeated inseries. Unless otherwise indicated, a homopolymeric segment can comprisean interruption in an otherwise consecutive series of nucleotides. Theinterruption can be 3 or fewer nucleotides differing from the othernucleotides making up the series. In some embodiments, the interruptionis a single nucleotide. An example of a homopolymeric segment comprisingan interruption is a first number of T residues, then one C residue, andthen a second number of T residues. Another example of a homopolymericsegment comprising an interruption is a first number of U residues, thenone C residue, and then a second number of U residues. Another exampleof a homopolymeric segment comprising an interruption is a first numberof A residues, then one G residue, and then a second number of Aresidues. The first and second numbers of A, T, or U residues in theforegoing examples can be, e.g., in the range of 5 to 10. In someembodiments, the first and second numbers of A, T, or U residues in theforegoing examples are in the range of 6 to 10. In some embodiments, thefirst and second numbers of A, T, or U residues in the foregoingexamples are in the range of 7 to 10. In some embodiments, the first andsecond numbers of A, T, or U residues in the foregoing examples are inthe range of 8 to 10. In some embodiments, the first and second numbersof A, T, or U residues in the foregoing examples are in the range of 9to 10. Alternatively, a homopolymeric segment can comprise a consecutiveseries of nucleotides (which is not interrupted).

The terms “variable length polymorphism”, “size polymorphism”, “repeatlength polymorphism” can be used interchangeably to refer topolymorphisms in the length of a segment at a given locus.

FIG. 1A depicts an exemplary workflow of a method disclosed herein. Themethod can be used for the assessment of a nucleic acid comprising ahomopolymeric segment. The method can comprise admixing a sample 100with a reaction solution 110 to create a reaction mixture. The reactionsolution 110 can be an aqueous solution. The sample 100 can be known orsuspected to comprise a nucleic acid comprising a homopolymeric segment.The method can further comprise subjecting the reaction mixture to areaction 120. The reaction 120 can comprise a nucleic acid synthesisreaction. The method can optionally further comprise performing ananalysis 130 of a reaction product generated by the reaction 120.

FIG. 1B depicts an exemplary reaction solution 110. The reactionsolution 110 can comprise NTPs 112. In some embodiments, the NTPscomprise dNTPs. In some embodiments, the NTPs comprise rNTPs. Thereaction solution 110 can further comprise a polymerase 114. Thereaction solution can further comprise one or more primers 116. Thereaction mixture can further comprise one or more additives 118.

The sample 100 can be a nucleic acid sample. The nucleic acid sample canbe any substance containing or presumed to contain nucleic acid. Thenucleic acid can be RNA, DNA, or any combination thereof. The DNA canbe, e.g., genomic DNA, mitochondrial DNA, viral DNA, synthetic DNA, orcDNA reverse transcribed from RNA. The RNA can be rRNA, tRNA, mRNA,siRNA, shRNA, miRNA, snoRNA, primary transcript RNA, or synthetic RNA. Anucleic acid in the sample can be fused to one or more nucleic acidadaptors. In some embodiments, the adaptors are heterologous. An adaptoris heterologous if fusion of the adaptor to the nucleic acid results ina non-naturally occurring sequence. The adaptors can be, e.g.,sequencing library adaptors or universal primer adaptors. The adaptorscan comprise one or more barcodes. In some cases, a nucleic acid in thesample need not be ligated to one or more adaptors.

The nucleic acid sample can be a biological sample. The nucleic acidsample can be an enriched nucleic acid sample. The enriched nucleic acidsample can be derived from a biological sample that has undergone apurification process. In some embodiments, the nucleic acid is purifiedfrom a biological sample, e.g., by a process which comprises removingone or more non-nucleic acid components from the biological sample. Thenucleic acid sample can comprise nucleic acid synthesized in vitro.Examples of in vitro nucleic acid synthesis include an amplificationreaction such as PCR, in vitro transcription, in vitro reversetranscription, in vitro primer extension, a sequencing reaction,phosphoramidite-based nucleic acid synthesis, and combinations thereof.

The biological sample can comprise liquid. It can be a fluid sample.Exemplary fluid biological samples include, e.g., whole blood, plasma,serum, soluble cellular extract, extracellular fluid, cerebrospinalfluid, ascites, urine, sweat, tears, saliva, buccal sample, a cavityrinse, or an organ rinse. The biological sample can comprise a solidsubstance, e.g., feces or tissue. Exemplary tissues include, e.g.,brain, bone, marrow, lung, heart, esophagus, stomach, duodenum, liver,prostate, nerve, meninges, kidneys, endometrium, cervix, breast, lymphnode, muscle, hair, and skin, among others. The biological sample can beobtained from a living subject, or can be obtained from a subjectpost-mortem. The biological sample can comprise cell culture, cells,and/or cell components. For example, the biological sample can comprisecell culture constituents, such as, e.g., cultured cells, conditionedmedia, recombinant cells, and cell components. In some embodiments, thebiological sample comprises cells. The cells can be primary cells, canbe immortalized cells from a cell line, can be mammalian, or can benon-mammalian (e.g., bacteria, yeast). The biological sample cancomprise a microbe, such as a virus, bacterium, protist, archaeon, orunicellular fungus. In some embodiments, the microbe is a virus. In someembodiments, the microbe is a bacterium. In some embodiments, thebiological sample comprises cell components.

The biological sample can be obtained from a subject. The subject can beany biological entity comprising genetic material. The subject can be ananimal, plant, fungus, or microorganism, such as, e.g., a bacterium,virus, archaeon, microscopic fungus, or protist. The subject can be amammal. The mammal can be a human.

In some embodiments, the subject is not diagnosed with a disease. Insome embodiments, the subject is diagnosed with a disease. In someembodiments, the subject is not suspected of being at risk for adisease. In some embodiments, the subject is suspected of being at riskfor a disease. The disease can be a degenerative disorder. Thedegenerative disorder can be a neurodegenerative disorder. In someembodiments, the neurodegenerative disorder is Alzheimer's disease.

In some embodiments, the sample 100 is known to harbor or suspected ofharboring a nucleic acid template. The nucleic acid template cancomprise one or more repeating A/T-rich segments, such as homopolymericsegments. The nucleic acid template can be known to comprise one or morerepeating A/T-rich segments, such as homopolymeric segments. The nucleicacid template can be suspected of comprising one or more repeatingA/T-rich segments, such as homopolymeric segments.

The homopolymeric segment can comprise consecutive T and/or U residues(wherein the segment can contain consecutive T residues, consecutive Uresidues, or consecutive residues that include both U and T residues),called a “T-homopolymeric segment.” The homopolymeric segment cancomprise consecutive residues which are either (i) A or (ii) T and/or Uresidues, but not both (i) and (ii). The homopolymeric segment cancomprise consecutive residues which are either (i) A or (ii) T residues,but not both (i) and (ii). The homopolymeric segment can compriseconsecutive residues which are (i) A or (ii) U residues, but not both(i) and (ii). The homopolymeric segment can comprise consecutive Aresidues, called an “A-homopolymeric segment.” The homopolymeric segmentcan comprise consecutive T residues. The homopolymeric segment cancomprise consecutive U residues.

The homopolymeric segment can comprise more than 10, more than 11, morethan 12, more than 13, more than 14, more than 15, more than 16, morethan 17, more than 18, more than 19, more than 20, more than 21, morethan 22, more than 23, more than 24, more than 25, more than 26, morethan 27, more than 28, more than 29, more than 30, more than 31, morethan 32, more than 33, more than 34, more than 35, more than 36, morethan 37, more than 38, more than 39, more than 40, more than 41, morethan 42, more than 43, more than 44, more than 45, more than 46, morethan 47, more than 48, more than 49, more than 50, more than 51, morethan 52, more than 53, more than 54, more than 55, more than 56, morethan 57, more than 58, more than 59, or more than 60 consecutiveresidues.

The homopolymeric segment can comprise a number of consecutive residuesranging from about 5 to about 40 consecutive residues. The homopolymericsegment can comprise a number of consecutive residues ranging from about10 to about 40 consecutive residues. The homopolymeric segment cancomprise a number of consecutive residues ranging from about 15 to about40 consecutive residues. The homopolymeric segment can comprise a numberof consecutive residues ranging from about 20 to about 40 consecutiveresidues. The homopolymeric segment can comprise a number of consecutiveresidues ranging from about 5 to about 50 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 10 to about 50 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 15 to about 50 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 20 to about 50 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 5 to about 60 consecutive residues. The homopolymericsegment can comprise a number of consecutive residues ranging from about10 to about 60 consecutive residues. The homopolymeric segment cancomprise a number of consecutive residues ranging from about 15 to about60 consecutive residues. The homopolymeric segment can comprise a numberof consecutive residues ranging from about 20 to about 60 consecutiveresidues. The homopolymeric segment can comprise a number of consecutiveresidues ranging from about 25 to about 40 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 5 to about 38 consecutive residues. The homopolymericsegment can comprise a number of consecutive residues ranging from about10 to about 38 consecutive residues. The homopolymeric segment cancomprise a number of consecutive residues ranging from about 15 to about38 consecutive residues. The homopolymeric segment can comprise a numberof consecutive residues ranging from about 20 to about 38 consecutiveresidues. The homopolymeric segment can comprise a number of consecutiveresidues ranging from about 25 to about 38 consecutive residues. Thehomopolymeric segment can comprise a number of consecutive residuesranging from about 5 to about 36 consecutive residues. The homopolymericsegment can comprise a number of consecutive residues ranging from about10 to about 36 consecutive residues. The homopolymeric segment cancomprise a number of consecutive residues ranging from about 15 to about36 consecutive residues. The homopolymeric segment can comprise a numberof consecutive residues ranging from about 20 to about 36 consecutiveresidues. The homopolymeric segment can comprise a number of consecutiveresidues ranging from about 25 to about 36 consecutive residues.

The nucleic acid template can be known to comprise or suspected ofcomprising a locus. The locus can comprise a repeating A/T-rich segment,such as a homopolymeric segment. The locus can be known to comprise orsuspected of comprising a polymorphism. The polymorphism can be avariable length polymorphism. The variable length polymorphism can be anA/T rich polymorphism. In some cases, the polymorphism is rs10524523.The locus can be in a gene. The gene can be associated with a disease.The disease can be a neurodegenerative disease. The neurodegenerativedisease can be Alzheimer's disease. The Alzheimer's disease can belate-onset Alzheimer's disease. In some cases, the gene is TOMM40. Insome cases, the locus is in intron 6 of TOMM40.

The NTPs 112 can comprise an AT/GC ratio. As used herein, an “AT/GCratio” can refer to a ratio of the total concentration of the sum ofnucleotide triphosphates comprising A, T, or U to the totalconcentration of the the sum of nucleotide triphosphates comprising G orC. For example, an “AT/GC” ratio can refer to a ratio of the totalconcentration of the sum of dATP, dUTP, and dTTP ([dATP]+[dUTP]+[dTTP])to the total concentration of the sum of dGTP and dCTP (e.g., can equal([dATP]+[dUTP]+[dTTP])/([dGTP]+[dCTP]). The AT/GC ratio can be biased,e.g., a ratio greater than 1. For example, the AT/GC ratio can be about2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, orhigher than 500.

The AT/GC ratio can be about 2 or higher, about 5 or higher, about 6 orhigher, about 7 or higher, about 8 or higher, about 9 or higher, about10 or higher, about 12.5 or higher, about 15 or higher, about 17.5 orhigher, about 20 or higher, about 25 or higher, about 30 or higher,about 35 or higher, about 40 or higher, about 45 or higher, about 50 orhigher, about 55 or higher, or about 60 or higher, about 70 or higher,about 80 or higher, about 90 or higher, about 100 or higher, about 120or higher, about 140 or higher, about 160 or higher, about 180 orhigher, about 200 or higher, about 250 or higher, about 300 or higher,about 350 or higher, about 400 or higher, about 450 or higher, or about500 or higher.

The AT/GC ratio can range from about 2 to about 25, range from about 2to about 60, range from about 5 to about 60, range from about 10 toabout 40, range from about 15 to about 30, range from about 5 to about25, range from about 8 to about 25, range from about 10 to about 25,range from about 15 to about 25, or range from about 18 to about 22. TheAT/GC ratio can range from a value of about X to about Y, wherein X andY have values described herein provided that Y is greater than X. X canbe 2. X can be 5. X can be 10. X can be 15. X can be 18. X can be 20. Xcan be 22. X can be 25. X can be 30. X can be 35. X can be 40. X can be45. X can be 50. X can be 55. X can be 60. X can be 70. X can be 80. Xcan be 90. X can be 100. X can be 120. X can be 140. X can be 160. X canbe 180. X can be 200. X can be 250. X can be 300. X can be 350. X can be400. X can be 450. Y can be 5. Y can be 10. Y can be 15. Y can be 18. Ycan be 20. Y can be 22. Y can be 25. Y can be 30. Y can be 35. Y can be40. Y can be 45. Y can be 50. Y can be 55. Y can be 60. Y can be 70. Ycan be 80. Y can be 90. Y can be 100. Y can be 120. Y can be 140. Y canbe 160. Y can be 180. Y can be 200. Y can be 250. Y can be 300. Y can be350. Y can be 400. Y can be 450. Y can be 500.

The NTPs 112 can comprise one of, more than one of, or all of a dNTPcomplementary to cytidine, a dNTP complementary to guanosine, a dNTPcomplementary to adenosine, and a dNTP complementary to thymidine. Forexample, the NTPs 112 can comprise one of, more than one of, or all ofdATP, dTTP, dCTP, dGTP, and dUTP. In some embodiments, the NTPs comprisedATP. dTTP, dCTP, and dGTP. In some embodiments, the NTPs comprise dATP.In some embodiments, the NTPs comprise a dNTP complementary to dATP. Insome embodiments, the NTPs comprise dCTP. In some embodiments, the NTPscomprise a dNTP complementary to dCTP. In some embodiments, the NTPscomprise dGTP. In some embodiments, the NTPs comprise a dNTPcomplementary to dGTP. In some embodiments, the NTPs comprise dTTP. Insome embodiments, the NTPs comprise a dNTP complementary to dTTP. Insome embodiments, the NTPs comprise dUTP. In some embodiments, the NTPscomprise a dNTP complementary to dUTP. In some embodiments, the NTPscomprise diaminopurine. In some embodiments, the NTPs comprise2-thiothymine. In some embodiments, the NTPs comprise 2-aminoadenine. Insome embodiments, the NTPs comprise at least one dideoxy-NTP (ddNTP). Insome embodiments, the NTPs comprise ddATP. In some embodiments, the NTPscomprise ddCTP. In some embodiments, the NTPs comprise ddGTP. In someembodiments, the NTPs comprise ddTTP. In some embodiments, the NTPscomprise ddUTP.

The NTP complementary to cytidine can be present at a concentration thatis about 5 μM or greater. The NTP complementary to cytidine can bepresent at a concentration that is about 10 μM or greater. The NTPcomplementary to cytidine can be present at a concentration that isabout 40 μM or greater. The NTP complementary to cytidine can be presentat a concentration that ranges from about 10 μM to about 400 μM. The NTPcomplementary to cytidine can be present at a concentration that rangesfrom about 40 μM to about 400 μM. For example, the NTP complementary tocytidine can be present at a concentration that is about 10 μM, about 20μM, about 30 μM, about 40 μM, about 41 μM, about 42 μM, about 43 μM,about 44 μM, about 45 μM, about 46 μM, about 47 μM, about 48 μM, about49 μM, about 50 μM, about 51 μM, about 52 μM, about 53 μM, about 54 μM,about 55 μM, about 56 μM, about 57 μM, about 58 μM, about 59 μM, about60 μM, about 61 μM, about 62 μM, about 63 μM, about 64 μM, about 65 μM,about 66 μM, about 67 μM, about 68 μM, about 69 μM, about 70 μM, about71 μM, about 72 μM, about 73 μM, about 74 μM, about 75 μM, about 76 μM,about 77 μM, about 78 μM, about 79 μM, about 80 μM, about 81 μM, about82 μM, about 83 μM, about 84 μM, about 85 μM, about 86 μM, about 87 μM,about 88 μM, about 89 μM, about 90 μM, about 91 μM, about 92 μM, about93 μM, about 94 μM, about 95 μM, about 96 μM, about 97 μM, about 98 μM,about 99 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM,about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM,about 325 μM, about 350 μM, about 375 μM, or about 400 μM.

The NTP complementary to guanosine can be present at a concentrationthat is about 5 μM or greater. The NTP complementary to guanosine can bepresent at a concentration that is about 10 μM or greater. The NTPcomplementary to guanosine can be present at a concentration that isabout 40 μM or greater. The NTP complementary to guanosine can bepresent at a concentration that ranges from about 10 μM to about 400 μM.The NTP complementary to guanosine can be present at a concentrationthat ranges from about 40 μM to about 400 μM. For example, the NTPcomplementary to guanosine can be present at a concentration that isabout 10 μM, about 20 μM, about 30 μM, about 40 μM, about 41 μM, about42 μM, about 43 μM, about 44 μM, about 45 μM, about 46 μM, about 47 μM,about 48 μM, about 49 μM, about 50 μM, about 51 μM, about 52 μM, about53 μM, about 54 μM, about 55 μM, about 56 μM, about 57 μM, about 58 μM,about 59 μM, about 60 μM, about 61 μM, about 62 μM, about 63 μM, about64 μM, about 65 μM, about 66 μM, about 67 μM, about 68 μM, about 69 μM,about 70 μM, about 71 μM, about 72 μM, about 73 μM, about 74 μM, about75 μM, about 76 μM, about 77 μM, about 78 μM, about 79 μM, about 80 μM,about 81 μM, about 82 μM, about 83 μM, about 84 μM, about 85 μM, about86 μM, about 87 μM, about 88 μM, about 89 μM, about 90 μM, about 91 μM,about 92 μM, about 93 μM, about 94 μM, about 95 μM, about 96 μM, about97 μM, about 98 μM, about 99 μM, about 100 μM, about 125 μM, about 150μM, about 175 μM, about 200 μM, about 225 μM, about 250 μM, about 275μM, about 300 μM, about 325 μM, about 350 μM, about 375 μM, or about 400μM.

In some cases, a NTP complementary to cytidine and a NTP complementaryto guanosine are both present at concentrations that are about 10 μM orgreater. In some cases, a NTP complementary to cytidine and a NTPcomplementary to guanosine are both present at concentrations that areabout 20 μM or greater. In some cases, a NTP complementary to cytidineand a NTP complementary to guanosine are both present at concentrationsthat are about 30 μM or greater. In some cases, a NTP complementary tocytidine and a NTP complementary to guanosine are both present atconcentrations that are about 40 μM or greater. In some cases, the NTPcomplementary to cytidine and NTP complementary to guanosine are bothpresent at concentrations that are between about 10 μM and 400 μM. Insome cases, the NTP complementary to cytidine and NTP complementary toguanosine are both present at concentrations that are between about 40μM and 400 μM.

The NTP complementary to adenosine can be present at a concentrationthat is about 20 μM or greater. For example, the NTP complementary toadenosine can be present at a concentration that is about 20 μM, about30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM,about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM,about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM,about 325 μM, about 350 μM, about 375 μM, about 400 μM, about 425 μM,about 450 μM, about 475 μM, about 500 μM, about 525 μM, about 550 μM,about 575 μM, about 600 μM, about 625 μM, about 650 μM, about 675 μM,about 700 μM, about 725 μM, about 750 μM, about 775 μM, about 800 μM,about 825 μM, about 850 μM, about 875 μM, about 900 μM, about 925 μM,about 950 μM, about 975 μM, about 1000 μM (1 mM), about 1.2 mM, about1.4 mM, about 1.6 mM, about 1.8 mM, about 2 mM, or higher. The NTPcomplementary to adenosine can be present at a concentration that rangesfrom about 20 μM and about 5 mM. The NTP complementary to adenosine canbe present at a concentration that ranges from about 50 μM and about 5mM. The NTP complementary to adenosine can be present at a concentrationthat ranges from about 100 μM and about 5 mM. The NTP complementary toadenosine can be present at a concentration that ranges from about 250μM and about 5 mM. The NTP complementary to adenosine can be present ata concentration that ranges from about 20 μM and about 3 mM. The NTPcomplementary to adenosine can be present at a concentration that rangesfrom about 50 μM and about 3 mM. The NTP complementary to adenosine canbe present at a concentration that ranges from about 100 μM and about 3mM. The NTP complementary to adenosine can be present at a concentrationthat ranges from about 250 μM and about 3 mM. The NTP complementary toadenosine can be present at a concentration that ranges from about 20 μMand about 2 mM. The NTP complementary to adenosine can be present at aconcentration that ranges from about 50 μM and about 2 mM. The NTPcomplementary to adenosine can be present at a concentration that rangesfrom about 100 μM and about 2 mM. The NTP complementary to adenosine canbe present at a concentration that ranges from about 250 μM and about 2mM. The NTP complementary to adenosine can be present at a concentrationthat ranges from about 700 μM and about 1.5 mM. The NTP complementary toadenosine can be present at a concentration that ranges from about 700μM and about 2 mM.

The NTP complementary to thymidine can be present at a concentrationthat is about 20 μM or greater. For example, the NTP complementary tothymidine can be present at a concentration that is about 20 μM, about30 μM, about 40 μM, about 50 μM, about 60 μM, about 70 μM, about 80 μM,about 90 μM, about 100 μM, about 125 μM, about 150 μM, about 175 μM,about 200 μM, about 225 μM, about 250 μM, about 275 μM, about 300 μM,about 325 μM, about 350 μM, about 375 μM, about 400 μM, about 425 μM,about 450 μM, about 475 μM, about 500 μM, about 525 μM, about 550 μM,about 575 μM, about 600 μM, about 625 μM, about 650 μM, about 675 μM,about 700 μM, about 725 μM, about 750 μM, about 775 μM, about 800 μM,about 825 μM, about 850 μM, about 875 μM, about 900 μM, about 925 μM,about 950 μM, about 975 μM, about 1000 μM (1 mM), about 1.2 mM, about1.4 mM, about 1.6 mM, about 1.8 mM, about 2 mM, or higher. The NTPcomplementary to thymidine can be present at a concentration that rangesfrom about 20 μM and about 5 mM. The NTP complementary to thymidine canbe present at a concentration that ranges from about 50 μM and about 5mM. The NTP complementary to thymidine can be present at a concentrationthat ranges from about 100 μM and about 5 mM. The NTP complementary tothymidine can be present at a concentration that ranges from about 250μM and about 5 mM. The NTP complementary to thymidine can be present ata concentration that ranges from about 20 μM and about 3 mM. The NTPcomplementary to thymidine can be present at a concentration that rangesfrom about 50 μM and about 3 mM. The NTP complementary to thymidine canbe present at a concentration that ranges from about 100 μM and about 3mM. The NTP complementary to thymidine can be present at a concentrationthat ranges from about 250 μM and about 3 mM. The NTP complementary tothymidine can be present at a concentration that ranges from about 20 μMand about 2 mM. The NTP complementary to thymidine can be present at aconcentration that ranges from about 50 μM and about 2 mM. The NTPcomplementary to thymidine can be present at a concentration that rangesfrom about 100 μM and about 2 mM. The NTP complementary to thymidine canbe present at a concentration that ranges from about 250 μM and about 2mM. The NTP complementary to thymidine can be present at a concentrationthat ranges from about 700 μM and about 1.5 mM. The NTP complementary tothymidine can be present at a concentration that ranges from about 700μM and about 2 mM.

In some cases, a NTP complementary to adenosine and a NTP complementaryto thymidine are both present at concentrations that are about 20 μM orgreater, about 50 μM or greater, about 150 μM or greater, about 200 μMor greater, about 250 μM or greater, about 500 μM or greater, about 750μM or greater, about 1000 μM or greater, about 2000 μM or greater, about3000 μM or greater or about 4000 μM or greater. In some cases, the NTPcomplementary to adenosine and NTP complementary to thymidine are bothpresent at concentrations that are between about 50 μM and about 4000μM. In some cases, the NTP complementary to adenosine and NTPcomplementary to thymidine are both present at concentrations that arebetween about 250 μM and about 2000 μM. In some cases, the NTPcomplementary to adenosine and NTP complementary to thymidine are bothpresent at concentrations that are between about 700 μM and 1500 μM. Insome cases, the NTP complementary to adenosine and NTP complementary tothymidine are both present at concentrations that are between about 700μM and 2000 μM.

The reaction solution 110 can comprise a total NTP concentration. Thetotal NTP concentration can be about 0.1 mM, about 0.2 mM, about 0.3 mM,about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM,about 0.9 mM, about 1 mM, about 1.2 mM, about 1.5 mM, about 2 mM, about2.1 mM, about 2.2. mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3 mM, about 3.1mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4 mM, about 4.1 mM,about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM,about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5 mM, about 5.1 mM,about 5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM,about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6 mM, about 6.1 mM,about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM,about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7 mM, about 7.1 mM,about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM,about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8 mM, about 8.1 mM,about 8.2 mM, about 8.3 mM, about 8.4 mM, about 8.5 mM, about 8.6 mM,about 8.7 mM, about 8.8 mM, about 8.9 mM, about 9 mM, about 9.1 mM,about 9.2 mM, about 9.3 mM, about 9.4 mM, about 9.5 mM, about 9.6 mM,about 9.7 mM, about 9.8 mM, about 9.9 mM, about 10 mM, about 10.1 mM,about 10.2 mM, about 10.3 mM, about 10.4 mM, about 10.5 mM, about 10.6mM, about 10.7 mM, about 10.8 mM, about 10.9 mM, or about 11 mM. In somecases, the total NTP concentration is about 2.1 mM. In some cases, thetotal NTP concentration is about 4.1 mM. In some cases, the total NTPconcentration is about 6.1 mM. In some cases, the total NTPconcentration is about 4.2 mM.

In some cases, the total NTP concentration ranges from about 0.4 mM toabout 8 mM. In some cases, the total NTP concentration ranges from about0.5 mM to about 5 mM. In some cases, the total NTP concentration rangesfrom about 2 mM to about 4.5 mM. In some cases, the total NTPconcentration ranges from about 2 mM to about 2.5 mM. In some cases, thetotal NTP concentration ranges from about 2.5 mM to about 3.5 mM. Insome cases, the total NTP concentration ranges from about 3.5 mM toabout 4.5 mM. In some cases, the total NTP concentration ranges fromabout 3.5 mM to about 4.2 mM.

The polymerase 114 can be a DNA polymerase. The DNA polymerase cancomprise a wild-type polymerase. The DNA polymerase can comprise amodified polymerase. The DNA polymerase can comprise a thermophilicpolymerase. The DNA polymerase can comprise a chimeric polymerase. TheDNA polymerase can comprise an engineered polymerase. The DNA polymerasecan comprise a mixture of more than one polymerase. Exemplary DNApolymerases include, e.g., a high-fidelity DNA polymerase (EXACTPOLYMERASE™ (5 PRIME GmbH), ACCUSURE™ DNA Polymerase (Bioline), PHUSION™ACCUPRIME™ Pfx (Invitrogen), Extensor Hi-Fidelity PCR Enzyme (ABgene),ACCUZYME™ DNA Polymerase (Bioline), OPTIMASE® DNA Polymerase(Transgenomic, Inc.), VELOCITY DNA Polymerase (Bioline), GENECHOICE®ACCUPOL™ DNA Polymerase (GeneChoice, Inc.), KOD HIFI™ DNA Polymerase(Novagen), EASY-A™ High-Fidelity PCR Cloning Enzyme (Stratagene), EXL™DNA Polymerase (Stratagene), KAPA HIFI™ DNA Polymerase (KapaBiosystems), HERCULASE® II Fusion DNA Polymerase (Stratagene),BIO-X-ACT™ Long DNA Polymerase (Bioline), BIO-X-ACT™ Short DNAPolymerase (Bioline), EU-Taq DNA Polymerase (EENZYME® LLC), PYROPHAGE®3173 DNA Polymerase, Pwo DNA Polymerase (Roche Applied Science), orPLATINUM Taq DNA Polymerase High Fidelity (Invitrogen)), a hot-start DNApolymerase (PHIRE™ Hot Start DNA Polymerase (New England Biolabs),PHUSION™ Hot Start High-Fidelity DNA Polymerase (New England Biolabs),JUMPSTART™ REDTAQ™ DNA Polymerase (Sigma-Aldrich), PFUULTRA™ HotstartDNA Polymerase (Stratagene), PFUTURBO® Cx Hotstart DNA Polymerase(Stratagene), PRIMESTAR™ HS DNA Polymerase (Takara), HotMaster™ Taq DNAPolymerase (5 PRIME GmbH), HOTTAQ™ DNA Polymerase (Abnova Corporation),AMPLITAQ GOLD® DNA Polymerase (Applied Biosystems), RED HOT DNAPolymerase (ABgene), ACCUPRIME™ GC-Rich DNA Polymerase (Invitrogen),PAQ5000™ DNA Polymerase (Stratagene), or SAHARA™ DNA Polymerase(Bioline)), a mixture of more than one polymerase (GENECHOICE® UNIPOL™DNA Polymerase (GeneChoice, Inc.), KOD XL™ DNA Polymerase (Novagen), LATAQ DNA Polymerase (Takara), EXPAND® 20 kb PLUS Thermostable DNApolymerase mixture (Roche Applied Science), EXPAND High Fidelity PLUSThermostable DNA polymerase mixture (Roche Applied Science), EXPAND HighFidelity Thermostable DNA polymerase mixture (Roche Applied Science),EXPAND® Long Template Thermostable DNA polymerase mixture (Roche AppliedScience), HERCULASE® Enhanced DNA Polymerase (Stratagene), KAPALONGRANGE™ DNA Polymerase (Kapa Biosystems), Synergy Taq DNA Polymerase(EENZYME® LLC), or ELONGASE® Enzyme Mix (Invitrogen)), a chimeric DNApolymerase (PFX50™ DNA Polymerase (Invitrogen), BIOLINE HYBRIPOL™ DNAPolymerase (Bioline), or PHUSION™ DNA Polymerase (New England Biolabs)),a modified DNA polymerase (KAPA2G™ Robust DNA Polymerase (KapaBiosystems), KAPA2G™ Robust HotStart DNA Polymerase (Kapa Biosystems),KAPA2G™ Fast DNA Polymerase (Kapa Biosystems), KAPA2G™ Fast HotStart DNAPolymerase (Kapa Biosystems), 9 DEGREES NORTH™ (Modified) DNA Polymerase(New England Biolabs), or THERMINATOR™ DNA Polymerase (New EnglandBiolabs)), an exo-DNA polymerase (Exo-Pfu DNA Polymerase (Stratagene),Bst DNA Polymerase Lg Frag (New England Biolabs), MASTERAMP™ Tfl DNAPolymerase (EPICENTRE Biotechnologies), Thermoprime Plus DNA Polymerase(ABgene), Taq-red DNA Polymerase (AppliChem GmbH), BIOTHERM™ Taq DNAPolymerase (EENZYME® LLC), GENECHOICE® REDPOL™ DNA Polymerase(GeneChoice, Inc.), or Exo Minus (Lucigen)), a high-yield DNA polymerase(YIELDACE™ DNA Polymerase (Stratagene) or E2TAK™ DNA Polymerase(Takara)), or naturally occurring DNA polymerases from P. kodakaraensis,P. furiosus, T. gorgonarius, T. zilligii, T. litoralis “Vent™”, P. GB-D“Deep Vent”, T. 9N-7, T. aggregans, T. barossii, T. fumicolans, T.celer, Pyrococcus sp. strain ST700, T. pacificus, P. abysii, T.profundus, T. siculi, T. hydrothermalis, Thermococcus sp. strain GE8, T.thioreducens, P. horikoshii or T. onnurineus NA1, Thermococcus sp. 9°N-7, Thermococcus sp. GI-J, Thermococcus sp. MAR-13, Thermococcus sp.GB-C, Thermococcus sp. GI-H, Thermus aquaticus, Thermus thermophilus,Thermus caldophilus, Thermus filiformis, Thermus flavus, Thermotogamaritima, Bacillus stearothermophilus, or Bacillus caldotenax, forexample. In certain embodiments, the DNA polymerase is Phoenix Hot StartTaq Polymerase®. In certain embodiments, the DNA polymerase isPhusion^(a) Hot Start High-Fidelity DNA Polymerase (New EnglandBiolabs). In certain embodiments, the DNA polymerase is Herculase® IIFusion DNA Polymerase (Stratagene).

The DNA polymerase can be a hot-start DNA polymerase. Exemplaryhot-start DNA polymerases include, e.g., Phoenix Hot Start TaqPolymerase® (Enzymatics), Phire™ Hot Start DNA Polymerase (New EnglandBiolabs), Phusion^(a) Hot Start High-Fidelity DNA Polymerase (NewEngland Biolabs), JumpStart™ REDTaq™ DNA Polymerase (Sigma-Aldrich),PfuUltra™ Hotstart DNA Polymerase (Stratagene), PfuTurbo® Cx HotstartDNA Polymerase (Stratagene), PrimeSTAR™ HS DNA Polymerase (Takara),among others. In some cases, the polymerase 114 is an RNA polymerase.

One or more primers 116 can prime polymerase-mediated extension into,across, or within a locus. The one or more primers 116 can hybridize toa template comprising the locus. The one or more primers 116 can amplifythe template comprising the locus. Exemplary loci are described herein.The locus can be known to harbor or suspected of harboring a segmentthat comprises one or more homopolymeric segments. Exemplaryhomopolymeric segments are described herein.

The one or more primers 116 can comprise a forward primer. The forwardprimer can anneal to a 5′ end of a template. For example, the forwardprimer can anneal to about 15-30, 15-25, 15-20, 20-30, or 20-25nucleotides at a 5′ end of the template. The one or more primers canalso comprise a reverse primer. The reverse primer can anneal to a 3′end of a template (e.g., to a 5′ end of a reverse strand of thetemplate). For example, the reverse primer can anneal to about 15-30,15-25, 15-20, 20-30, or 20-25 nucleotides at a 3′ end of the template.

The one or more primers 116 can comprise a first primer that hybridizesto a location upstream of a variable length polymorphism. In some cases,a portion of the first primer can hybridize to a portion of the variablelength polymorphism. The one or more primers 116 can comprise a secondprimer that hybridizes to a location downstream of the variable lengthpolymorphism. In some cases, a portion of the second primer canhybridize to a portion of the variable length polymorphism. The secondprimer can hybridize to a portion of the variable length polymorphismthat is smaller than the smallest known allele of the variable lengthpolymorphism. For instance, if the smallest known allele of a variablelength polymorphism of interest is 10 nucleotides, the second primer canhybridize to 9 or fewer nucleotides of the variable length polymorphism.In some cases, a first or second primer can comprise a 3′-terminalsequence that preferentially hybridizes to about 4 to about 9consecutive A or T residues.

The variable length polymorphism can be a TOMM40 polymorphism. In someembodiments, the variable length polymorphism is the rs10524523polymorphism.

In some embodiments, the one or more primers 116 comprises a firstprimer that hybridizes to a location upstream of a variable lengthpolymorphism, such as within 500, 300, 200, 100, or 50 nucleotides ofthe variable length polymorphism. In some embodiments, the first primerspecifically hybridizes to a location separated from a variable lengthpolymorphism by 1 to about 500 nucleotides. In some embodiments, thefirst primer specifically hybridizes to a location separated from avariable length polymorphism by 1 to about 300 nucleotides. In someembodiments, the first primer specifically hybridizes to a locationseparated from a variable length polymorphism by 1 to about 200nucleotides. In some embodiments, the first primer specificallyhybridizes to a location separated from a variable length polymorphismby 1 to about 100 nucleotides. In some embodiments, the first primerspecifically hybridizes to a location separated from a variable lengthpolymorphism by 1 to about 50 nucleotides. In some embodiments, thefirst primer specifically hybridizes to a location separated from avariable length polymorphism by about 50 to about 500 nucleotides. Insome embodiments, the first primer specifically hybridizes to a locationseparated from a variable length polymorphism by about 50 to about 300nucleotides. In some embodiments, the first primer specificallyhybridizes to a location separated from a variable length polymorphismby about 50 to about 200 nucleotides. In some embodiments, the firstprimer specifically hybridizes to a location separated from a variablelength polymorphism by about 50 to about 100 nucleotides. The one ormore primers 116 can comprise a second primer that hybridizes to alocation downstream of the variable length polymorphism, such as within500, 300, 200, 100, or 50 nucleotides of the variable lengthpolymorphism. In some embodiments, the second primer specificallyhybridizes to a location separated from a variable length polymorphismby 1 to about 500 nucleotides. In some embodiments, the second primerspecifically hybridizes to a location separated from a variable lengthpolymorphism by 1 to about 300 nucleotides. In some embodiments, thesecond primer specifically hybridizes to a location separated from avariable length polymorphism by 1 to about 200 nucleotides. In someembodiments, the second primer specifically hybridizes to a locationseparated from a variable length polymorphism by 1 to about 100nucleotides. In some embodiments, the second primer specificallyhybridizes to a location separated from a variable length polymorphismby 1 to about 50 nucleotides. In some embodiments, the second primerspecifically hybridizes to a location separated from a variable lengthpolymorphism by about 50 to about 500 nucleotides. In some embodiments,the second primer specifically hybridizes to a location separated from avariable length polymorphism by about 50 to about 300 nucleotides. Insome embodiments, the second primer specifically hybridizes to alocation separated from a variable length polymorphism by about 50 toabout 200 nucleotides. In some embodiments, the second primerspecifically hybridizes to a location separated from a variable lengthpolymorphism by about 50 to about 100 nucleotides. The variable lengthpolymorphism can be a TOMM40 polymorphism. In some embodiments, thevariable length polymorphism is the rs10524523 polymorphism. Specifichybridization to a location means that a primer preferentially binds tothat location over other locations in the sample under a primer bindingcondition suitable for a nucleic acid synthesis reaction, such as asolution comprising 50 mM KCl, pH 8, at one or more hybridizationtemperatures such as 42° C., 45° C., 50° C., 55° C., 56° C., 57° C., 58°C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67°C., 68° C., 69° C., 70° C., 71° C., or 72° C.

Exemplary primers include, e.g., CCAAAGCATTGGGATTACTGGC (primer 001)(SEQ ID NO: 1) and GATTGCTTGAGCCTAGGCATTC (primer 002) (SEQ ID NO: 2).In some embodiments, primer 001 is detectably labeled. In someembodiments, primer 001 is detectably labeled with a fluorophore. Insome embodiments, primer 001 is detectably labeled with FAM. In someembodiments, primer 002 is detectably labeled. In some embodiments,primer 002 is detectably labeled with a fluorophore. In someembodiments, primer 002 is detectably labeled with FAM.

In some embodiments, the one or more primers 116 comprises a set ofprimers. The set of primers can comprise at least one primer describedherein. In some embodiments, the set of primers is capable of primingpolymerase-mediated extension into more than one locus. In someembodiments, the set of primers comprises primers collectively capableof hybridizing specifically to a plurality of templates that comprise orare suspected of harboring a locus of interest. In some embodiments, theset of primers is capable of amplifying a plurality of templatescomprising a plurality of loci of interest.

The one or more additives 118 can comprise a buffer. Exemplary buffersinclude, e.g., tris(hydroxymethyl)aminomethane (Tris), bis-tris propane,bicarbonate, phosphate, glycine, histidine,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),3-(N-morpholino)propanesulfonic acid (MOPS), and various conjugatebases/acids and salts thereof.

The one or more additives 118 can comprise magnesium (Mg). The termmagnesium, as used herein, includes magnesium in solution (solute andsolvated/hydrated forms) and in its ionized forms. The magnesium can bein the form of a magnesium salt, including solute and solvated/hydratedforms, e.g., magnesium ions and counterions in solution. The magnesiumsalt can be a chemical compound containing magnesium and the conjugatebase of an acid. Exemplary magnesium salts include, without limitation,magnesium chloride, magnesium acetate, magnesium sulfate, magnesiumbromide, or magnesium iodide. The magnesium salts can be provided insuch quantity that the final concentration of magnesium can be in agiven range. In some embodiments, the magnesium concentration rangesfrom about 1 to about 11 mM. In some embodiments, the magnesiumconcentration ranges from about 1 to about 10 mM. In some embodiments,the magnesium concentration ranges from about 1 to about 7.5 mM. In someembodiments, the magnesium concentration ranges from about 1 to about 5mM. In some embodiments, the magnesium concentration ranges from about 1to about 4.5 mM. In some embodiments, the magnesium concentration rangesfrom about 1 to about 4 mM. In some embodiments, the magnesiumconcentration ranges from about 1 to about 3.5 mM. In some embodiments,the magnesium concentration ranges from about 1 to about 3 mM. In someembodiments, the magnesium concentration ranges from about 1.5 to about3 mM. In some embodiments, the magnesium concentration ranges from about2 to about 5 mM. In some embodiments, the magnesium concentration rangesfrom about 2 to about 11 mM. In some embodiments, the magnesiumconcentration ranges from about 2 to about 10 mM. In some embodiments,the magnesium concentration ranges from about 2 to about 7.5 mM. In someembodiments, the magnesium concentration ranges from about 2.5 to about11 mM. In some embodiments, the magnesium concentration ranges fromabout 2.5 to about 10 mM. In some embodiments, the magnesiumconcentration ranges from about 2.5 to about 7.5 mM. In someembodiments, the magnesium concentration ranges from about 2.5 to about5 mM. In some embodiments, the magnesium concentration ranges from about3 to about 11 mM. In some embodiments, the magnesium concentrationranges from about 3 to about 10 mM. In some embodiments, the magnesiumconcentration ranges from about 3 to about 7.5 mM. In some embodiments,the magnesium concentration ranges from about 3 to about 5 mM. In someembodiments, the magnesium concentration ranges from about 1.5 to about4.5 mM. In some embodiments, the magnesium concentration ranges fromabout 2 to about 4 mM. For example, the final concentration of magnesiumcan be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, or 11mM. In some embodiments, the magnesium concentration ranges from about 1mM to 7 mM more than the total NTP concentration. In some embodiments,the magnesium concentration ranges from about 1 mM to 6 mM more than thetotal NTP concentration. In some embodiments, the magnesiumconcentration ranges from about 1 mM to 5 mM more than the total NTPconcentration. In some embodiments, the magnesium concentration rangesfrom about 1 mM to 4 mM more than the total NTP concentration. In someembodiments, the magnesium concentration ranges from about 1 mM to 3 mMmore than the total NTP concentration. In some embodiments, themagnesium concentration ranges from about 1 mM to 2 mM more than thetotal NTP concentration. In some embodiments, the magnesiumconcentration ranges from about 1 mM to 1 mM more than the total NTPconcentration. The magnesium can be present at a molarity that rangesfrom about 70% to about 300% of the molarity of total NTPs. Themagnesium can be present at a molarity that ranges from about 80% toabout 300% of the molarity of total NTPs. The magnesium can be presentat a molarity that ranges from about 70% to about 250% of the molarityof total NTPs. The magnesium can be present at a molarity that rangesfrom about 80% to about 250% of the molarity of total NTPs. Themagnesium can be present at a molarity that ranges from about 70% toabout 200% of the molarity of total NTPs. The magnesium can be presentat a molarity that ranges from about 80% to about 200% of the molarityof total NTPs. The magnesium can be present at a molarity that rangesfrom about 70% to about 150% of the molarity of total NTPs. Themagnesium can be present at a molarity that ranges from about 80% toabout 150% of the molarity of total NTPs. The magnesium can be presentat a molarity that ranges from about 90% to about 125% of the molarityof total NTPs.

The one or more additives 118 can comprise one or more enhancers. Insome cases, the one or more enhancers comprises one or more of betaine,DMSO, and a neutral detergent. In some cases, the one or more enhancerscomprises one or more of betaine, a betaine analog, DMSO, and a neutraldetergent. “Betaine” refers to N,N,N-trimethylglycine. A “betaineanalog” is any neutral chemical compound with a positively chargedcationic functional group which bears no hydrogen atom, for example, anammonium ion or phosphonium ion, and with a negatively chargedfunctional group such as a carboxylate group which may not be adjacentto the cationic site. In some embodiments, the betaine analog has amolecular weight less than or equal to about 600 Da. The betaine analogcan have a molecular weight less than or equal to about 300 Da. Thebetaine analog can have a molecular weight ranging from about 75 toabout 600 Da. The betaine analog can have a molecular weight rangingfrom about 75 to about 300 Da. Additionally or alternatively, thebetaine analog can comprise an ammonium moiety and/or a carboxylatemoiety. The one or more additives 118 can comprise betaine and/or abetaine analog. Betaine and/or a betaine analog can be present at amolar concentration ranging from 0.01 to 5 M, 0.01 to 4 M, 0.01 to 3 M,0.01 to 2.5 M, 0.02 to 5 M, 0.03 to 5 M, 0.04 to 5 M, 0.05 to 5M, 0.07to 5M, 0.1 to 5 M, 0.2 to 5 M, 0.3 to 5 M, 0.4 to 5 M, 0.5 to 5 M, 0.7to 5 M, 1 to 5 M, 1.5 to 5M, 0.1 to 4 M, 0.5 to 3 M, 0.5 to 2.5 M, or0.5 to 2.5 M, for example, about 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 0.75,1, 1.25, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5,4, 4.5, or 5 M. In some cases, the one or more additives 118 compriseabout 1 M betaine.

The one or more additives 118 can comprise DMSO. DMSO can be present inthe reaction solution at a concentration that ranges from about 0.1% toabout 10% (v/v). DMSO can be present in the reaction solution at aconcentration that ranges from about 0.5% to about 5%. DMSO can bepresent in the reaction solution at a concentration that ranges fromabout 0.5% to about 10%. DMSO can be present in the reaction solution ata concentration that ranges from about 0.1% to about 5%. DMSO can bepresent in the reaction solution at a concentration that ranges fromabout 0.5% to about 3%. The DMSO can be present in the reaction solutionat a concentration that is about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 3.3%, about3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about4%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.7%, about5.8%, about 5.9%, about 6%, about 6.1%, about 6.2%, about 6.3%, about6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, about7%, about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about8.8%, about 8.9%, about 9%, about 9.1%, about 9.2%, about 9.3%, about9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%, about 9.9%, orabout 10%. In some cases, the one or more additives 118 comprise about1% DMSO. In some cases, the one or more additives 118 comprise about 2%DMSO.

In some cases, the reaction solution comprises two enhancers. In somecases, the reaction solution comprises three enhancers. In some cases,the reaction solution comprises betaine and DMSO. In some cases, thereaction solution comprises a betaine analog and DMSO. In some cases,the reaction solution comprises betaine, DMSO, and a neutral detergent.Exemplary concentrations of betaine and DMSO are disclosed herein.

Other additives that can be present in the reaction solution include,but are not limited to, non-specific background/blocking nucleic acids(e.g., salmon sperm DNA), biopreservatives (e.g. sodium azide), andinhibitors (e.g. RNAse inhibitors).

Some embodiments of a reaction solution 110 comprise a NTPs 112 with anAT/GC ratio of about 2 or greater. In some embodiments, the reactionsolution comprises about 100 μM each dATP and dTTP, and 50 μM each dGTPand dCTP. In some embodiments, the NTPs 112 have an AT/GC ratio of about5. In some embodiments, the reaction solution comprises about 250 μMeach dATP and dTTP, and 50 μM each dGTP and dCTP. In some embodiments,the NTPs 112 have an AT/GC ratio of about 10. In some embodiments, thereaction solution comprises about 500 μM each dATP and dTTP, and 50 μMeach dGTP and dCTP. In some embodiments, the NTPs 112 have an AT/GCratio of about 20. For example, the reaction solution can comprise about1000 μM each dATP and dTTP, and 50 μM each dGTP and dCTP. In someembodiments, the reaction solution comprises about 2000 μM each dATP anddTTP, and 100 μM each dGTP and dCTP.

The reaction solution can further comprise about 1.5 mM to about 4 mMMg²⁺. In some embodiments, the reaction solution comprises about 2.5 mMMg²⁺. In some embodiments, the reaction solution comprises about 2 mMMg²⁺. In some embodiments, the reaction solution comprises about 4 mMMg²⁺. The reaction solution can further comprise betaine. The betainecan be present in a molarity that ranges from about 0.1 to 2 M betaine,for example, between about 0.5 to about 1.5 M betaine. In someembodiments, the betaine is present at a 1 M concentration. The reactionsolution can further comprise DMSO. The DMSO can be present at aconcentration that can be between about 0.1% and about 10%, for example,between about 0.5% and about 4%.

Table 1 below lists exemplary embodiments of reaction solutionsdisclosed herein.

TABLE 1 Exemplary embodiments of reaction solutions 110 EmbodimentEmbodiment Embodiment Embodiment Component 1 2 3 4 [dATP], 1000, 10001000, 1000 2000, 2000 2000, 2000 [dTTP] (μM) [dGTP], 50, 50 50, 50 50,50 100, 100 [dCTP] (μM) Mg²⁺ 2.5 2 4 4 Betaine (M) 0 or 1 0 or 1 0 or 10 or 1 DMSO (%) 0 or 1 0 or 1 0 or 1 0 or 1

In some cases, the reaction solution comprises one or more labelssuitable for labeling a reaction product. In some embodiments, one ormore labels are covalently attached to one or more primers. In someembodiments, a primer comprises a covalently attached label. In someembodiments, one or more labels are covalently attached to one or morenucleotide triphosphates. In some embodiments, a nucleotide triphosphatecomprises a covalently attached label. Labels include, but are notlimited to: light-emitting, light-scattering, and light-absorbingcompounds which generate or quench a detectable fluorescent,chemiluminescent, or bioluminescent signal (see, e.g., Kricka, L.,Nonisotopic DNA Probe Techniques, Academic Press, San Diego (1992) andGarman A., Non-Radioactive Labeling, Academic Press (1997)).

In some embodiments, a fluorophore is used as a label. Fluorophoresuseful as labels include, but are not limited to, fluoresceins (see,e.g., U.S. Pat. Nos. 5,188,934, 6,008,379, and 6,020,481), rhodamines(see, e.g., U.S. Pat. Nos. 5,366,860, 5,847,162, 5,936,087, 6,051,719,and 6,191,278), benzophenoxazines (see, e.g., U.S. Pat. No. 6,140,500),coumarins, energy-transfer fluorescent dyes, comprising pairs of donorsand acceptors (see, e.g., U.S. Pat. Nos. 5,863,727; 5,800,996; and5,945,526), cyanines (see, e.g., WO 9745539), lissamines,phycoerythrins, pyrenyloxytrisulfonic acid-based fluorophores (e.g.,Cascade Blue®), and any derivatives thereof. Examples of fluoresceindyes include, but are not limited to, Fluorescein Isothiocyanate(“FITC”); 6-carboxyfluorescein (“FAM”); 5-Tetrachloro-Fluorescein,(“TET”); 2′,4′,1,4,-tetrachlorofluorescein;2′,4′,5′,7′,1,4-hexachlorofluorescein;6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (“HEX”); fluorinatedanalogs of fluorescein (such as Oregon Green® 488, Oregon Green® 500,and Oregon Green® 514); and6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (“JOE”). Exemplarycyanine dyes include, without limitation, Cy2, Cy3, Cy3.5, Cy5, Cy5.5,Cy7, and the WellRed® infrared dyes D1, D2, D3 and D4. Exemplaryrhodamine dyes include, e.g., Rhodamine Green, Rhodamine Red,Tetramethylrhodamine, carboxytetramethylrhodamine (“TAMRA”),sulforhodamine 101 acid chloride (Texas Red), and carboxy-X-rhodamine(“ROX”). Exemplary coumarin dyes include, e.g.,6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific Blue™) and aminomethylcoumarin acetate (“AMCA”). Additional labels can be derivedfrom, e.g., FluorX (Amersham). Fluorophores can include Alexa Fluor®dyes (e.g., sulfonated versions of dye molecules such as, withoutlimitation, fluorescein, rhodamine, cyanine, coumarin, and the like).Exemplary Alexa Fluor® dyes include, e.g., Alexa 350, Alexa 430, Alexa430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594.Fluorophores can include BODIPY™ dyes (comprising the core structure4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), such as, e.g., BODIPY630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX.In certain aspects, the fluorescent label is selected from fluorescentlabels that are compatible with CE analysis such as FAM, TET, ROX, NED™,VIC™, or JOE.

In some embodiments, the label is a radioactive label. The radioactivelabel can be ³²P. The radioactive label can be ³³P. The radioactivelabel can be ³⁵S. In some embodiments, the label is an electrochemicallabel. The electrochemical label can be ferrocene. In some embodiments,the label is an affinity label. The affinity label can be biotin. Theaffinity label can be digoxygenin.

The reaction solution can comprise more than one label. In someembodiments, the reaction solution comprises different fluorophorescapable of emitting light at different, spectrally-resolvablewavelengths (e.g., 4 differently colored fluorophores); certain suchlabeled probes are known in the art and described above, and in U.S.Pat. No. 6,140,054. A dual labeled fluorescent probe that includes areporter fluorophore and a quencher fluorophore is used in someembodiments. Other examples can include Freedom® dyes that arecommercially available surrogates for common dyes. It will beappreciated that pairs of fluorophores can be chosen to have distinctemission spectra so that they can be easily distinguished.

The reaction 120 (see FIG. 1A) can comprise a nucleic acid synthesisstep. The nucleic acid synthesis step can comprise annealing the one ormore primers 116 to a nucleic acid template. The nucleic acid synthesisstep can further comprise polymerase-mediated extension of the one ormore primers 116 along the template. In some embodiments, the one ormore primers 116 are extended into a locus of interest. In someembodiments, the one or more primers 116 are extended within a locus ofinterest. In some embodiments, the one or more primers 116 are extendedacross a locus of interest. Exemplary loci are described herein. In somecases, the reaction 120 generates reaction products which are subjectedto analysis 130. For example, polymerase-mediated extension of the oneor more primers 116 can generate extension products which are subjectedto analysis 130.

The reaction 120 can comprise an amplification reaction. Theamplification reaction can generate amplification products (e.g.,amplicons). The amplification products can be subjected to analysis 130.Examples of amplification reactions include, without limitation, PCR,NASBA (nucleic acid sequence based amplification), SDA (stranddisplacement amplification), LAMP (loop-mediated isothermalamplification), and RCA (rolling circle amplification). See, e.g., U.S.Pat. No. 4,683,202 (PCR); U.S. Pat. No. 6,326,173 and Journal ofVirological Methods 151:283-293 (2008) (NASBA); U.S. Pat. No. 5,648,211(SDA); U.S. Pat. No. 6,410,278 (LAMP); and U.S. Pat. No. 6,287,824(RCA). All of the foregoing are incorporated herein by reference. Theskilled artisan will understand what reagents are appropriate toprovide. Each of these methods involves DNA synthesis, and as suchinvolves the use of DNA polymerases, nucleotides, and divalent cations(supplied as a salt), particularly magnesium, in a solution conducive toDNA polymerization and in which the template is present. The methods canvary in terms of providing additional catalytic activities, the use ofthermocycling or isothermal incubation, and the use and structure ofprimers. A buffer at a suitable pH is also typically provided. In someembodiments, the suitable pH ranges from about 7 to about 8. In someembodiments, the suitable pH ranges from about 6.5 to about 8.5. In someembodiments, the suitable pH ranges from about 6 to about 9. In someembodiments, the suitable pH ranges from about 7.4 to about 7.5.

In some cases, the reaction 120 comprises PCR. PCR can comprise repeatedrounds of amplification. A “round” or “cycle” of amplification cancomprise a denaturation step, a primer annealing step, and apolymerase-mediated extension step. The reaction can be thermocycled soas to drive denaturation of nucleic acids in a high temperature step,annealing of the primers to templates at a lower temperature step, andextension at a temperature which can be but is not necessarily higherthan that of the annealing step. In some cases, the PCR comprises anannealing step at a temperature at or below 75° C. In some cases, thePCR comprises an annealing step at a temperature at or below 70° C. Insome cases, the PCR comprises an annealing step at a temperature at orbelow 65° C. In some cases, the PCR comprises an annealing step at atemperature that ranges from about 50° C. to about 75° C. In some cases,the PCR comprises an annealing step at a temperature that ranges fromabout 57° C. and about 63° C. In some cases, the PCR comprises anannealing step at a temperature that ranges from about 52° C. and about58° C. In some cases, the PCR comprises an annealing step at atemperature that ranges from about 62° C. and about 68° C. Amplificationcan proceed as the amplification products of one cycle can serve astemplate in the next cycle. Amplification can proceed in a linear orexponential fashion. In linear PCR, the reaction mixture can compriseone or more forward primers to be extended into, within, or across aregion of interest. In exponential PCR, the reaction mixture cancomprise forward and reverse primers which flank a region of interest.In some embodiments, a touchdown annealing procedure is used. In atouchdown annealing procedure, a first annealing temperature is used inan early cycle, such as the first cycle, and a second annealingtemperature, lower than the first annealing temperature, is used a latercycle later than the early cycle. The touchdown annealing procedure cancomprise using a lower annealing temperature than in the previous cyclein one or more cycles. The touchdown annealing procedure can compriseusing a lower annealing temperature than in the previous cycle in 1 to20 cycles. The touchdown annealing procedure can comprise using a lowerannealing temperature than in the previous cycle in 1 to 15 cycles. Thetouchdown annealing procedure can comprise using a lower annealingtemperature than in the previous cycle in 1 to 10 cycles. The touchdownannealing procedure can comprise using a lower annealing temperaturethan in the previous cycle in 5 to 20 cycles. The touchdown annealingprocedure can comprise using a lower annealing temperature than in theprevious cycle in 5 to 15 cycles. The touchdown annealing procedure cancomprise using a lower annealing temperature than in the previous cyclein 5 to 10 cycles. The touchdown annealing procedure can comprise usinga lower annealing temperature than in the previous cycle in 10 to 20cycles. The touchdown annealing procedure can comprise using a lowerannealing temperature than in the previous cycle in 10 to 15 cycles. Insome embodiments, the touchdown annealing procedure comprises a cyclewith a first annealing temperature ranging from about 58° C. to about72° C. In some embodiments, the touchdown annealing procedure comprisesa cycle with a first annealing temperature ranging from about 60° C. toabout 70° C. In some embodiments, the touchdown annealing procedurecomprises a cycle with a first annealing temperature ranging from about62° C. to about 68° C. In some embodiments, the touchdown annealingprocedure comprises a cycle with a first annealing temperature rangingfrom about 64° C. to about 66° C. In some embodiments, the touchdownannealing procedure comprises a cycle with a second annealingtemperature ranging from about 48° C. to about 62° C. In someembodiments, the touchdown annealing procedure comprises a cycle with asecond annealing temperature ranging from about 50° C. to about 60° C.In some embodiments, the touchdown annealing procedure comprises a cyclewith a second annealing temperature ranging from about 52° C. to about58° C. In some embodiments, the touchdown annealing procedure comprisesa cycle with a second annealing temperature ranging from about 54° C. toabout 56° C. The second annealing temperature can be lower than thefirst annealing temperature. The second annealing temperature can beused in a cycle later that the cycle in which the first annealingtemperature is used, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 cycles after the cycle in which the firstannealing temperature is used. When the second annealing temperature isused in a cycle more than one cycle after the first annealingtemperature is used, the annealing temperatures in the intervening cycleor cycles can be about the same as the first annealing temperature.Alternatively, the annealing temperatures in the intervening cycle orcycles can be about the same as the second annealing temperature.Alternatively, the annealing temperatures in the intervening cycle orcycles can be or between the first and the second annealing temperature.For example, the annealing temperature in an intervening cycle or cyclescan decrease linearly. The rate of linear decrease can range from, e.g.,0.2° C. per cycle to 10° C. per cycle. The rate of linear decrease canrange from 0.5° C. per cycle to 5° C. per cycle. The rate of lineardecrease can range from 0.5° C. per cycle to 3° C. per cycle. The rateof linear decrease can range from 0.5° C. per cycle to 2° C. per cycle.The rate of linear decrease can range from 0.5° C. per cycle to 1.5° C.per cycle. The rate of linear decrease can range from 0.7° C. per cycleto 1.3° C. per cycle.

The reaction 120 can comprise between about 1 to about 40 amplificationcycles. For example, the reaction 120 can comprise between about 10 toabout 40 cycles. For example, the reaction 120 can comprise betweenabout 15 to about 35 cycles. The reaction 120 can comprise about 2,about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10,about 11, about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, about 20, about 21, about 22, about 23, about 24,about 25, about 26, about 27, about 28, about 29, about 30, about 31,about 32, about 33, about 34, about 35, about 36, about 37, about 38,about 39, or about 40 amplification cycles. In some cases, the reaction120 comprises no more than 35 amplification cycles. In some cases, thereaction 120 comprises no more than 30 amplification cycles. In somecases, the reaction 120 comprises no more than 25 amplification cycles.

In NASBA, an RNA polymerase (RNAP) is provided in addition to the DNApolymerase, which can also be a reverse transcriptase (RT) (e.g., anenzyme that can catalyze DNA synthesis using either an RNA or DNAtemplate). Primers can be provided that are similar to those used in PCRexcept that at least one primer can additionally comprise a promotersequence that is recognized by the RNAP. Thus, the product of the RTserves as template for the RNAP, which synthesizes RNA that serves astemplate for the RT, leading to amplification. In some forms of NASBA,RNase H is provided to produce single-stranded DNA after synthesis of anRNA-DNA hybrid by RT. Amplification occurs via the combined action ofthe RT and RNAP, in the absence of repeated thermal denaturation.

SDA is a technique in which DNA is amplified in an isothermal andasynchronous manner, meaning that cyclic thermal denaturation is notused to separate the strands; instead, strand displacement occursthrough DNA synthesis itself, wherein extension of a 3′ OH causesdisplacement of the downstream strand. The 3′ OH is provided initiallyby an exterior primer and subsequently by a nicking reaction. Two pairsof primers can be provided. One ‘interior’ pair binds surrounding theamplicon and additionally comprises 5′ flaps containing a restrictionsite. The other, ‘exterior’ pair is positioned distally, i.e., furtherfrom the target region. An interior primer can bind the template, beextended, and then be displaced by synthesis from the correspondingexterior primer. Subsequently, the displaced DNA is madedouble-stranded, e.g., by second strand synthesis. The next step is tonick one strand of the double stranded molecule, which can be done byusing modified nucleotides and a restriction site wherein the cleavagesite is inactivated on one strand (but not the other) by the modifiednucleotide. The restriction enzyme corresponding to this site isprovided in the reaction and generates the nick. The 3′ OH at theresulting nick is then extended by the DNA polymerase, displacing onestrand (which can again serve as a template) and the regenerated doublestrand molecule is again a substrate for nicking followed by extensionand displacement, leading to amplification. Repeated thermaldenaturation is not necessary.

LAMP is an amplification procedure designed to be highly specific, thatis, it can discriminate between templates differing by only a singlenucleotide polymorphism (SNP), in that one allele is a substrate foramplification and the other is not. It is also isothermal. As in SDA,two pairs of primers, interior and exterior, can be provided; theinterior primers can also have a 5′ flap. However, in LAMP, the 5′ flapof each interior primer contains a sequence matching a sequence withinthe template strand to which it binds, interior to the site where the 3′portion of the primer binds. For example, if the primer anneals to the(+) strand of a template molecule, which contains the downstreamsequence A, then the primer flap can also contain sequence A. Notably,the SNP locus which is to be discriminated by this reaction is locatedat the edge of the region bound by the flap, corresponding to the lastbase at the 5′ end of the flap. The last base at the 5′ end of thereverse interior primer flap also corresponds to the SNP locus. As inSDA, the interior primer is extended and then displaced by extension ofthe exterior primer. When this occurs, the 5′ flap forms a loop bybinding its complement (which is now part of the same molecule;continuing the above example, the displaced strand contains the reversecomplement of sequence A, designated sequence T, and the sequence A inthe flap binds intramolecularly to sequence T). The reverse interiorprimer anneals to the looped displaced strand, interior to its 3′ end(which corresponds to the reverse exterior primer) and primes synthesis,which displaces the loop and forms a partially double-stranded,partially single stranded DNA. Then, a reverse exterior primer annealsto the single stranded portion and primes synthesis, causing stranddisplacement. The displaced strand can now form a loop wherein its 3′end is paired to an internal portion of the molecule. Only if the SNPlocus matches the 3′ end (which is derived from an interior primer flapthat was exogenously supplied) does extension occur. Further primerannealing, looping, and extension/displacement events, described in thereference cited above, result in selective amplification of templateswith the SNP allele matching the primer flap.

In RCA, a circular DNA template is used. A primer anneals to the circleand is extended continuously, with the polymerase displacing the DNAsynthesized during the previous revolution as it proceeds. This reactionproceeds with linear kinetics and produces long, concatemerizedproducts. In double-primed RCA, a second primer is provided that annealsto the concatemerized product of the above reaction. This version of thereaction allows use of product as template, and therefore results inexponential kinetics. As in other isothermal reactions, product is madesuitable for annealing to primer in double-primed RCA through stranddisplacement due to extension of upstream primers; in this case theprimers can be bound to other concatemers further upstream in thetemplate strand.

Reaction products generated by a reaction 120 can be subjected toanalysis 130 (see FIG. 1A). The analysis can be useful for assessment ofgenomic regions comprising repeating A/T rich segments and homopolymericsegments, such as homopolymeric segments of A, T, or U residues, whichcan be consecutive or interrupted once by one to three othernucleotides.

In some cases, the analysis comprises determining a sequence of areaction product. The term “determining a sequence” as used hereinrefers to a method by which the identities of at least 8 consecutivenucleotides of a polynucleotide are obtained. In some embodiments, theidentities of at least 10 consecutive nucleotides of a polynucleotideare obtained. In some embodiments, the identities of at least 12consecutive nucleotides of a polynucleotide are obtained. In someembodiments, the identities of at least 16 consecutive nucleotides of apolynucleotide are obtained. In some embodiments, the sequence of ahomopolymeric segment is determined by comparing capillaryelectrophoresis data for an amplification reaction product generatedfrom a sample comprising the homopolymeric segment. The capillaryelectrophoresis data can be interpreted by comparing it to calibrationdata from one or more standards. The one or more standards can compriseone or more amplification reaction products generated from a referencesample. The reference sample can comprise a nucleic acid having a knownsequence. The reference sample can comprise an artificially synthesizednucleic acid. The reference sample can comprise a nucleic acidcontaining a homopolymeric segment of a known length. Sequencing methodscan comprise a sequencing reaction that creates or modifies nucleic acidmolecules artificially so as to make them detectable, thereby allowingsequence determination. Sequencing methods can comprise using asequencing apparatus which can detect a nucleic acid molecule in anelectromagnetic manner, e.g., based on electromagnetic radiation (e.g.,fluorescent or radioactive emission) or electromagnetic field effects(as in, e.g., nanopore sequencing). Sequencing methods suitable foranalysis of reaction products described herein can include Sangersequencing or next-generation sequencing. Next generation sequencing caninvolve sequencing of clonally amplified DNA templates or single DNAmolecules in a massively parallel fashion. Exemplary sequencing methodsinclude, but are not limited to, sequencing-by-synthesis, ionpyrosequencing, reversible dye terminator sequencing, semiconductorsequencing, sequencing by ligation, single-molecule sequencing,sequencing by hybridization, and nanopore sequencing. Platforms forsequencing by synthesis can include those available from IIlumina, 454Life Sciences, Helicos Biosciences, Thermo Fisher/Ion Torrent (e.g.,Personal Genome Machine, Proton), and Oxford Nanopore (eg, MinION) andQiagen. Exemplary Illumina platforms are described in Gudmundsson et al(Nat. Genet. 2009 41:1122-6), Out et al (Hum. Mutat. 2009 30:1703-12)and Turner (Nat. Methods 2009 6:315-6), U.S. Patent ApplicationPublication No. US20080160580, U.S. Pat. Nos. 6,306,597 and 7,115,400.Exemplary Helicos Biosciences platforms include the True Single MoleculeSequencing platform. Exemplary platforms for ion semiconductorsequencing are described in U.S. Pat. No. 7,948,015 and include, e.g.,the Ion Torrent Personal Genome Machine (PGM). Exemplary platforms forpryosequencing are described in U.S. Pat. Nos. 7,211,390; 7,244,559; and7,264,929, and can include the GS Flex. Exemplary platforms forsequencing by ligation are described in U.S. Pat. No. 5,750,341 andinclude, e.g., the SOLiD sequencing platform. Exemplary platforms forsingle-molecule sequencing include the Helicos True Single MoleculeSequencing platform and the SMRT® system from Pacific Biosciences. Insome cases, extension products are subjected to nucleic acid sequencing,without requiring amplification prior to sequencing. For example,sequencing by, e.g., the SMRT® system by Pacific Biosciences cancomprise sequencing an extension product described herein as it is beingsynthesized.

In some cases, analysis 130 comprises size analysis. Size analysis cancomprise determining the size of one or more reaction products generatedby a method described herein. Size analysis can comprise determining anamount of reaction products having a certain size.

Size analysis can comprise an electrophoresis method. Exemplaryelectrophoresis methods include, e.g., gel electrophoresis and capillaryelectrophoresis (CE). In some cases, size analysis comprises CEanalysis. CE analysis can comprise use of instrumentation such as theABI 3100, 3130, 3730, or 3500 models. Other implementations include anyinstrument capable of electrophoretic sizing of DNA and multicolorresolution. For example, the Beckman Vidiera or SEQ6000 capillaryelectrophoresis systems for the detection of WellRed infrared dyes (D1,D2, D3 and D4) can also be used, or the Li-Cor instrument incorporatingIRDyes. Other methods that can be used include microfluidic CE systemssuch as the Agilent 2100 Bioanalyzer and similar platforms, massspectrometry, agarose gel electrophoresis followed by scan densitometry,and analysis of radiolabeled products using phosphorImager or scandensitometry of autoradiographs. In some cases, size analysis comprisesassessing intensities of peaks observed in CE electropherograms,phosphorimager scans, densitometric scans, mass spectra, or other formsof data. Size analysis can comprise determination of peak height, areaunder the curve (integration), or curve fitting. In some embodiments,size analysis comprises comparing data from a sample to data from one ormore standards with one or more repeating nucleotide segments of knownlength. In some embodiments, size analysis comprises comparing data froma sample to data from one or more standards with one or more repeatingNT rich segments or homopolymeric nucleotide segments of known length.The comparison can comprise regression analysis. An example of using ofstandards with similar AT/GC content to extrapolate a length of anucleic acid segment is provided in Filipovic-Sadic S, et al., “A novelFMR1 PCR method for the routine detection of low abundance expandedalleles and full mutations in fragile X syndrome,” Clin Chem. 2010March; 56(3):399-408 (doi: 10.1373/clinchem.2009.136101, Epub 2010January 7). This approach can be adapted for use with repeating A/T richsegments or homopolymeric segments, among others.

Methods described herein can be used to determine a length of arepeating A/T rich segment or homopolymeric segment, such as ahomopolymeric segment of A, T, or U residues, which can be consecutiveor interrupted once by one to three other nucleotides.

Methods described herein can detect a repeating A/T rich segment orhomopolymeric segment above 10 nucleotides in length, which can beconsecutive or interrupted once by one to three other nucleotides. Forexample, methods described herein can detect A- or T-homopolymericsegments or repeating A/T rich segments that are above 8, above 9, above10, above 11, above 12, above 13, above 14, above 15, above 16, above17, above 18, above 19, above 20, above 21, above 22, above 23, above24, above 25, above 26, above 27, above 28, above 29, above 30, above31, above 32, above 33, above 34, above 35, above 36, above 37, above38, above 39, above 40, above 41, above 42, above 43, above 44, above45, above 46, above 47, above 48, above 49, above 50, above 51, above52, above 53, above 54, above 55, above 56, above 57, above 58, above59, or above 60 nucleotides in length. Methods described herein candetect A- or T-homopolymeric segments or repeating A/T rich segmentsthat range from about 10 to about 40 nucleotides in length. Methodsdescribed herein can detect A- or T-homopolymeric segments or repeatingA/T rich segments that range from about 10 to about 50 nucleotides inlength. Methods described herein can detect A- or T-homopolymericsegments or repeating A/T rich segments that range from about 10 toabout 48 nucleotides in length. Methods described herein can detect A-or T-homopolymeric segments or repeating A/T rich segments that rangefrom about 10 to about 60 nucleotides in length. Methods describedherein can detect A- or T-homopolymeric segments or repeating A/T richsegments that range from about 8 to about 60 nucleotides in length.Methods described herein can detect A- or T-homopolymeric segments orrepeating A/T rich segments that range from about 15 to about 40nucleotides in length. Methods described herein can detect A- orT-homopolymeric segments or repeating NT rich segments that range fromabout 20 to about 40 nucleotides in length. Methods described herein candetect A- or T-homopolymeric segments or repeating A/T rich segmentsthat range from about 30 to about 40 nucleotides in length. Methodsdescribed herein can detect A- or T-homopolymeric segments or repeatingA/T rich segments that range from about 15 to about 50 nucleotides inlength. Methods described herein can detect A- or T-homopolymericsegments or repeating A/T rich segments that range from about 20 toabout 50 nucleotides in length. Methods described herein can detect A-or T-homopolymeric segments or repeating A/T rich segments that rangefrom about 30 to about 50 nucleotides in length. Methods describedherein can detect A- or T-homopolymeric segments or repeating A/T richsegments that range from about 15 to about 48 nucleotides in length.Methods described herein can detect A- or T-homopolymeric segments orrepeating A/T rich segments that range from about 20 to about 48nucleotides in length. Methods described herein can detect A- orT-homopolymeric segments or repeating NT rich segments that range fromabout 30 to about 48 nucleotides in length. Methods described herein candetect A- or T-homopolymeric segments or repeating A/T rich segmentsthat range from about 15 to about 60 nucleotides in length. Methodsdescribed herein can detect A- or T-homopolymeric segments or repeatingA/T rich segments that range from about 20 to about 60 nucleotides inlength. Methods described herein can detect A- or T-homopolymericsegments or repeating A/T rich segments that range from about 30 toabout 60 nucleotides in length.

Methods described herein can be used to detect and distinguish aplurality of repeat length polymorphisms in a single sample or across aplurality of samples. For example, methods described herein candistinguish amplicons containing A- or T-homopolymeric segments orrepeating A/T rich segments that are below 20 nucleotides in length,between 20 and 29 nucleotides in length, and 30 or more nucleotides inlength. In some cases, methods described herein can distinguishamplicons containing A- or T-homopolymeric segments or repeating A/Trich segments that differ in size by 1 nucleotide. In some cases,methods described herein can distinguish amplicons containing A- orT-homopolymeric segments or repeating A/T rich segments that differ insize by 2 nucleotides or less. In some cases, methods described hereincan distinguish amplicons containing A- or T-homopolymeric segments orrepeating NT rich segments that differ in size by 3 nucleotides or less.In some cases, methods described herein can distinguish ampliconscontaining A- or T-homopolymeric segments or repeating A/T rich segmentsthat differ in size by 4 nucleotides or less. In some cases, methodsdescribed herein can distinguish amplicons containing A- orT-homopolymeric segments or repeating A/T rich segments that differ insize by 5 nucleotides or less. In some cases, methods described hereincan distinguish amplicons containing A- or T-homopolymeric segments orrepeating A/T rich segments that differ in size by 6 nucleotides orless. In some cases, methods described herein can distinguish ampliconscontaining A- or T-homopolymeric segments or repeating A/T rich segmentsthat differ in size by 7 nucleotides or less. In some cases, methodsdescribed herein can distinguish amplicons containing A- orT-homopolymeric segments or repeating A/T rich segments that differ insize by 8 nucleotides or less. In some cases, methods described hereincan distinguish amplicons containing A- or T-homopolymeric segments orrepeating A/T rich segments that differ in size by 9 nucleotides orless. In some cases, methods described herein can distinguish ampliconscontaining A- or T-homopolymeric segments or repeating NT rich segmentsthat differ in size by 10 nucleotides or less. In some embodiments, themethods described herein can distinguish amplicons containing A- orT-homopolymeric segments or repeating A/T rich segments that are atleast 20 nucleotides long and that differ in size by a number ofnucleotides as discussed above. In some embodiments, methods describedherein can distinguish amplicons containing A- or T-homopolymericsegments or repeating A/T rich segments that are at least 30 nucleotideslong and that differ in size by a number of nucleotides as discussedabove.

In some cases, methods described herein are capable of distinguishing afirst sample comprising a first template with a homopolymeric segment oflength (n+1) and a second template with a homopolymeric segment oflength (n−1) from a sample comprising a template with a homopolymericsegment of length (n), wherein n is greater than about 20 and less thanabout 40. In some cases, n is greater than about 30 and less than about40. In some cases, n is greater than about 35 and less than about 40. Insome cases, methods described herein are capable of distinguishing afirst sample comprising a first template with a repeating A/T richsegment of length (n+1) and a second template with a repeating A/T richsegment of length (n−1) from a sample comprising a template with arepeating A/T rich segment of length (n), wherein n is greater thanabout 20 and less than about 40. In some cases, n is greater than about30 and less than about 40. In some cases, n is greater than about 35 andless than about 40.

For example, methods described herein can be capable of distinguishing afirst sample comprising true 34 T and 36 T alleles of an rs10524523locus from a sample comprising non-target 35 T segments which resultfrom polymerase slippage/stutter. FIG. 2 depicts an exemplary desiredpeak profile from CE analysis of a sample known to be heterozygous forthe 34 T/36 T alleles. In a desired peak profile, one or both of the 34T and 36 T peaks have a greater intensity than the intensity of anon-target 35 T peak. By contrast, in an exemplary undesired peakprofile, a non-target 35 T peak intensity is higher than intensity ofthe 34 T and 36 T peaks.

Disclosed herein are kits useful for performing one or more methodsdescribed herein. A kit can comprise NTPs, such as dNTPs, having anAT/GC ratio greater than 2. A kit can include individual aliquots ofNTPs and instructions for preparing a reaction solution comprising NTPsas described herein. A kit can further comprise a primer suitable forextension into, within, or across a homopolymeric segment of at least 10consecutive A or T residues. A kit can further comprise a primersuitable for extension into, within, or across a repeating A/T richsegment of at least 10 consecutive A or T residues. In some cases, a kitcomprises at least two primers. The primers can be suitable foramplifying a genetic locus comprising a homopolymeric segment of atleast 10 consecutive A residues or at least 10 consecutive T residues.In some cases, the homopolymeric segment is the rs10524523 polymorphismof the TOMM40 gene. In some cases, a kit comprises at least one primerthat hybridizes upstream of the rs10524523 polymorphism of the TOMM40gene. In some cases, a kit comprises a second primer that hybridizesdownstream of the rs10524523 polymorphism of the TOMM40 gene. In somecases, the AT/GC ratio has a value described herein, such as a valuethat ranges from about 10 to about 40 or a value that ranges from about15 and about 30. In some cases, a kit further comprises a polymerase,which can be a polymerase described herein. In some cases, a kit furthercomprises magnesium in a molar amount in a range disclosed herein, e.g.,in the range from about 80% to about 150% of the molar amount of totalNTPs. In some embodiments, a kit further comprises a magnesium stocksolution. In some embodiments, a kit further comprises a solutioncomprising magnesium in a concentration of about 10 mM to about 2.5 M.In some embodiments, a kit further comprises a solution comprisingmagnesium in a concentration of about 10 mM to about 3 M. In some cases,a kit further comprises one or more additives, which can be one or moreadditives described herein. Exemplary additives are described herein. Insome cases, a kit further comprises betaine. In some cases, a kitfurther comprises a betaine analog. In some cases, a kit furthercomprises DMSO. In some cases, a kit further comprises betaine and DMSO.

A kit can include reference standards. The reference standards cancomprise one or more repeating A/T-rich segments, such as one or morehomopolymeric segments, of known lengths. Exemplary homopolymericsegments are described herein.

A kit can include a packaging material. As used herein, the term“packaging material” can refer to a physical structure housing thecomponents of the kit. The packaging material can maintain sterility ofthe kit components, and can be made of material commonly used for suchpurposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules,etc.). A kit can also include a buffering agent, a preservative, or aprotein/nucleic acid stabilizing agent.

Methods and kits described herein can have many applications. Forexample, methods described herein can be useful in disease diagnosis,disease prediction, selection of a therapeutic regimen, genotyping,identification, forensics, nucleic acid profiling, kinship analysis,genetic linkage analysis, marker-assisted selection, assessment of generegulation, population genetics, and taxonomic studies, among others.

Methods and kits described herein can be useful for of detecting agenotype associated with late-onset Alzheimer's disease. For example,methods described herein can be useful for genotyping a rs10524523polymorphism of the TOMM40 gene.

EXAMPLES

The following examples illustrate various embodiments and are notintended to limit the scope of the invention.

Example 1. Effect of Biased dNTP Ratios on PolymeraseSlippage/Stuttering

Heterozygous DNA samples, each containing two known poly-T repeatlengths for the TOMM40 poly-T polymorphism, were provided as describedin Table 2.

TABLE 2 Sample names and poly-T repeat lengths. rs10524523 Repeat lengthgenotype Sample Name (nt/nt) RS1310 35/36 RS1311 16/36 RS1319 34/36

10 ng of sample DNA were amplified using Phoenix Taq (Enzymatics) in aPCR reaction mixture comprising 1× Phoenix buffer and 2.5 mM Mg2+. Theprimers were /56-FAM/CCAAAGCATTGGGATTACTGGC (SEQ ID NO: 1) (Forward) andGATTGCTTGAGCCTAGGCATTC (SEQ ID NO: 2) (Reverse). The following finalconcentrations of dNTPs were used in the reactions: 250 μM each dNTP;100 μM each of dATP and dTTP with 50 μM each of dCTP and dGTP (“100/50”AT/GC ratio); 250 μM each of dATP and dTTP with 50 μM each of dCTP anddGTP (“250/50”), 500 μM each of dATP and dTTP with 50 μM each of dCTPand dGTP (“500/50”), and 1000 μM each of dATP and dTTP with 50 μM eachof dCTP and dGTP (“1000/50”). PCR was conducted for 35 cycles and thecrude product was diluted 100-fold prior to capillary electrophoresis(CE) analysis. CE analysis was performed on an ABI 3500×L. PCR productswere injected at 2.5 kv for 20 seconds. ROX 400HD was used as ladder.Mobility and correction factor (as described in Filipovic-Sadic et al.,2010, supra) were obtained from standards of known polyT length (8 T, 12T, 16 T, 20 T, 24 T, 48 T) and extrapolated to determine the genotype ofthe unknown sample.

FIGS. 3A-3D depict CE peak profiles for each DNA sample listed in Table2, with the target peaks labeled. Because the poly-T lengths are knownfor each sample, polymerase slippage/stuttering can be quantified by thenumber of extra non-target peaks and/or by the ratio of the target peak(N) height to one or more non-target peak heights, such as the height ofthe N−1 non-target peak. For example, a reduction in the number ofnon-target peaks (e.g., that exceed a threshold relative to the targetor maximum peak) or a higher target peak height as compared to one ormore non-target peak heights can be used, separately or together, toassess whether slippage/stuttering is reduced.

CE peak profiles from the RS1310 samples (35 T/36 T) are depicted inFIG. 3A. Peak profiles from the RS1310 samples demonstrate that,compared to the non-biased dNTP ratio, there was an increase in theratios of heights of the 35 T and 36 T target peaks to non-target peakheights at 250/50 AT/GC. The increase in the peak height ratios for the35 T and 36 T target peaks were more pronounced for the 500/50 AT/GC and1000/50 AT/GC reaction products. For the 1000/50 AT/GC ratio, theheights of the 35 T and 36 T peaks increased, the heights of thenon-target peaks decreased, and fewer non-target peaks were apparent, ascompared to the non-biased dNTP ratio of 250/250 AT/GC.

CE peak profiles from the RS1311 samples (16 T/36 T) are depicted inFIGS. 3B and 3C. Peak profiles from the RS1311 samples demonstrate that,compared to the non-biased dNTP ratio, the 500/50 and 1000/50 AT/GCreactions products exhibited an increase in the target/non-target peakheight ratios for the 16 T and 36 T target peaks, with the greatestincrease apparent for the 1000/50 ratio. There was also a reduction innon-target peak number and intensity relative to the results when thenon-biased dNTP ratio of 250/250 AT/GC was used.

CE peak profiles from the RS1319 samples (34 T/36 T) are depicted inFIG. 3D. The relative height of the non-target 35 T peak as compared totarget 34 T and 36 T peak heights can be used to indicate polymeraseslippage/stuttering errors, with shorter 35 T peak heights generallyindicating reduced slippage/stuttering. FIG. 3C demonstrates that the 35T peak was shortest relative to the target 34 T and 36 T peaks at the1000/50 AT/GC ratio.

These results, taken together, demonstrate that AT/GC ratios such as,e.g., 250/50, 500/50, and 1000/50 reduced polymerase slippage andstuttering, and improved A or T homopolymer repeat length analysis.

Example 2: Effect of AT/GC Ratios, Mg²⁺ Concentration and Total dNTPConcentration on Polymerase Slippage/Stutter

The effects of AT/GC ratio, Mg²⁺ concentration, and total dNTPconcentration on polymerase slippage/stuttering were measured byconducting a series of reactions with varying AT/GC concentrations(250/250, 500/500, 1000/1000, 2000/2000, 1000/50, 2000/50, 3000/50, and2000/100), varying MgSO₄ concentration (2, 2.5, 4, 6, 8, and 10 mM), andvarying total dNTP concentration (1-8 mM). All reaction mixtures wereadmixed with 10 ng of RS1319 sample DNA (34 T/36 T). PCR was conductedfor 27 cycles. CE analysis was performed as described in Example 1.

Results are depicted in FIGS. 4 and 5. FIG. 4 depicts results of varyingMg²⁺ concentration and total dNTP concentration when AT/GC ratios werenot biased. FIG. 4 confirmed that excess dNTP concentrations relative toMg²⁺ can inhibit DNA polymerase by sequestering magnesium ions.

FIG. 5 depicts results from varying Mg²⁺ concentration and total dNTPconcentration under different AT/GC ratio conditions. As shown in FIG.5, the non-target 35 T peak height was shorter than at least one of the34 T and 36 T target peaks for multiple reaction conditions including:1000/50 AT/GC, 2 mM Mg²+; 1000/50 AT/GC, 2.5 mM Mg²+; 1000/50 AT/GC, 4mM Mg²+; 2000/50 AT/GC, 4 mM Mg²⁺; and 2000/100 AT/GC, 4 mM Mg²⁺.

Example 3: Effect of Betaine and DMSO on Polymerase Slippage/Stuttering

To further evaluate reaction conditions for analysis of A- or T-richhomopolymeric regions, the effects of AT/GC ratio, betaine, and DMSOwere assessed by conducting a series of reactions with varying AT/GCratios (250/250, 250/25, 500/25, 500/50, and 1000/50), varying betaineamounts (0M, 1M), and varying DMSO concentrations (0%, 1%, 2%, and 4%).All reaction mixtures were admixed with 10 ng of RS1311 sample DNA (16T/36 T). PCR was conducted for 35 cycles. CE analysis was performed asdescribed in Example 1.

Results are depicted in FIGS. 6 and 7. FIG. 6 depicts the effects ofDMSO and betaine titration in the vicinity of the 16 T peak. Peakstutter generally decreased with biased AT/GC ratio conditions ascompared to unbiased AT/GC ratio conditions, with the greatest decreaseapparent at the 1000/50 ratio. The presence of 1M Betaine improvedstutter, including in unbiased AT/GC ratio conditions.

FIG. 7 depicts the effects of DMSO and betaine titration in the vicinityof the 36 T peak. Peak stutter generally decreased with biased AT/GCratio conditions as compared to unbiased AT/GC ratio conditions, withthe greatest decrease apparent at the 1000/50 ratio. 1M Betaine and 1%DMSO were also beneficial in reducing the stutter ratio (N−1/N).

Example 4: Effect of Reducing Number of PCR Cycles on PolymeraseStutter/Slippage

The effect of lowering number of PCR cycles on polymeraseslippage/stutter was assessed by varying PCR cycle numbers (25, 30, or35 cycles). 10 ng of either RS1311 (16 T/36 T) or RS1319 (34 T/36 T) DNAsamples were admixed with a PCR reaction mixture containing an AT/GCratio of 1000/50, 1% DMSO, 1M Betaine, and 2.0 mM MgSO₄. PCR was run for25, 30, or 35 cycles prior to CE analysis.

Results are depicted in FIGS. 8 and 9. FIG. 8 depicts the effect of PCRcycle numbers on 16 T and 36 T peak stutter. Because RS1311 samples areknown to contain 16 T and 36 T alleles in a 1:1 ratio, 36/16 peak heightratios that approach 1 indicate reduced bias toward amplification of theshorter target allele. FIG. 8 shows that among the PCR cycle numberstested, 25 cycles of PCR resulted in the 36/16 peak height ratio closestto 1.

FIG. 9 depicts the effect of PCR cycle numbers on 34 T and 36 T peakstutter. As shown in FIG. 9, the 35 T non-target peak was shorterrelative to the 34 T and 36 T peak heights with 25 or 30 PCR cycles ascompared to 35 PCR cycles, with the shortest height demonstrated under25 PCR cycle conditions.

Example 5: Combination of Biased dNTPs, Betaine, DMSO, and Lowered PCRCycles Improved Polymerase Slippage/Stutter

Heterozygous DNA samples described in Table 2 were provided. Sampleswere subjected to PCR reaction conditions as shown in Table 3.

TABLE 3 Reaction conditions. A B AT/GC concentration 250/250 1000/50ratio (μM/μM) Mg++ 2.5 mM MgCl₂ 2.0 mM MgSO₄ Betaine (M) 0 1 DMSO (%) 01 PCR cycles 35 27

Results are depicted in FIGS. 10-12. FIG. 10 depicts results from theRS1311 (16 T/36 T) samples. As compared to the condition A, theincreased AT/GC concentration ratio, lowered PCR cycle number, 1MBetaine, and 1% DMSO in condition B increased target/non-target peakheight ratios for the 16 T and 36 T target peaks, and also increased the36 T/16 T peak height ratio closer to 1.

FIG. 11 depicts results from the RS1310 (35 T/36 T) samples. As comparedto condition A, the increased AT/GC concentration ratio, lowered PCRcycle number, 1M Betaine, and 1% DMSO in condition B increasedtarget/non-target peak height ratios for the 35 T and 36 T target peaks,and reduced the number of non-target peaks.

FIG. 12 depicts results from the RS1319 samples. As compared tocondition A, the increased AT/GC concentration ratio, lowered PCR cyclenumber, 1M Betaine, and 1% DMSO in condition B reduced the 35 Tnon-target peak height relative to the 34 T and 36 T peak heights.

Example 6: Comparison of Conditions with and without DMSO and Betaine

Samples were subjected to PCR reaction conditions and analyzed bycapillary electrophoresis as follows.

TABLE 4 Reaction conditions. B C AT/GC concentration 1000/50 1000/50ratio (μM/μM) Mg++ 2.0 mM MgSO₄ 2.0 mM MgSO₄ Betaine (M) 1 0 DMSO (%) 10

For both conditions, MgSO₄ was supplied via Phoenix Hot Start Taqbuffer. The PCR program was 95° C. 5 min; 10× (95° C., 30 s; 65° C. to56° C. touchdown 1° C. per cycle, 30 s; 64° C., 30 s); 17× (95° C., 30s; 55° C., 60 s).

FIG. 13A shows products amplified from RS1310 (35 T/36 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds. The n−1/nratio for the 34 T and 35 T peaks was 0.46.

FIG. 13B shows products amplified from RS1310 (35 T/36 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds. Then−1/n ratio for the 34 T and 35 T peaks was 0.38.

FIG. 14A shows products amplified from RS1311 (16 T/36 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds. The n−1/nratio for the 35 T and 36 T peaks was 0.49. The peak height ratio of the36 T peak to the 16 T peak was 0.57.

FIG. 14B shows products amplified from RS1311 (16 T/36 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds. Then−1/n ratio for the 35 T and 36 T peaks was 0.46. The peak height ratioof the 36 T peak to the 16 T peak was 0.49.

FIG. 15A shows products amplified from RS1317 (29 T/36 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds. The n−1/nratio for the 28 T and 29 T peaks was 0.42.

FIG. 15B shows products amplified from RS1317 (29 T/36 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds. Then−1/n ratio for the 28 T and 29 T peaks was 0.34.

FIG. 16A shows products amplified from RS1318 (16 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds.

FIG. 16B shows products amplified from RS1318 (16 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds.

FIG. 17A shows products amplified from RS1319 (34 T/36 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds. The n−1/nratio for the 33 T and 34 T peaks was 0.45. The peak height ratio of the35 T peak to the 34 T peak was 0.88.

FIG. 17B shows products amplified from RS1319 (34 T/36 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds. Then−1/n ratio for the 33 T and 34 T peaks was 0.39. The peak height ratioof the 35 T peak to the 34 T peak was 0.86.

FIG. 18A shows products amplified from NA07541 (34 T/38 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds.

FIG. 18B shows products amplified from NA07541 (34 T/38 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds.

FIG. 19A shows products amplified from NA20243 (16 T/20 T) samples usingcondition C. The products were loaded at 2.5 kV for 5 seconds. The n−1/nratio for the 15 T and 16 T peaks was 0.26.

FIG. 19B shows products amplified from NA20243 (16 T/20 T) samples usingcondition B. The products were loaded at 2.5 kV for 20 seconds. Then−1/n ratio for the 15 T and 16 T peaks was 0.16.

The foregoing results generally show that condition C gave greateramplification efficiency, in that compared to condition B, four-foldlower load amounts (2.5 kV for 5 seconds) of products from condition Cgave target peak heights similar to or moderately lower (less than 50%decrease) than peak heights for condition B products loaded at 2.5 kVfor 20 seconds. The results also show that condition B permitted greatertarget vs. non-target peak discrimination in that, e.g., the n−1/n peakheight ratios were generally lower. Furthermore, the results fromcondition C show that betaine and DMSO are not essential. Thus,conditions B and C illustrate how reaction conditions can be tailored tofocus on amplification efficiency or target vs. non-target peakdiscrimination. High amplification efficiency can facilitateamplification procedures with reduced cycle numbers, and it was shownabove that reduced cycle numbers can improve target vs. non-target peakdiscrimination.

Example 7: Amplification of 48 T Homopolymeric Segment

A sample of a synthetic DNA template containing a 48 T homopolymericsegment was amplified using conditions A and B as in Example 5 andanalyzed by capillary electrophoresis. Results from condition A areshown in FIG. 20A. Results from condition B are shown in FIG. 20B. Thehighest peak in FIG. 20B represents the 48 T target peak. Non-targetpeaks containing 49 T, 50 T, 51 T, 52 T, and 53 T segments were alsodetectable. Thus, homopolymeric segments of 48 nucleotides or more canbe amplified and analyzed according to this disclosure.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims. The listing ofsteps in a method in a particular order is not to be construed as anindication that the steps must be performed in that order, except wherethere is an explicit indication to the contrary or the result of onestep is required for occurrence of another step.

1-73. (canceled)
 74. A kit comprising: at least two primers; and NTPs inan AT/GC ratio of 8 or higher, wherein the at least two primers arecapable of amplifying at least one DNA template comprising: (i) ahomopolymeric segment of at least 10 consecutive A residues or at least10 consecutive T residues; or (ii) a repeating A/T-rich segmentcomprising (T_(n)A)_(m), (AT_(n))_(m), (TA_(n))_(m), or (A_(n)T)_(m),wherein n is 2 or greater and m is such that the length of the repeatingNT-rich segment is 10 or more residues; wherein the at least one DNAtemplate further comprises target and non-target nucleic acid sequences.75. The kit of claim 74, wherein the at least two primers comprise atleast one primer comprising a covalently attached label.
 76. The kit ofclaim 74, wherein the at least two primers comprise a first primer thathybridizes upstream of the rs10524523 variable length polymorphism ofthe TOMM40 gene and a second primer that hybridizes downstream of thers10524523 variable length polymorphism of the TOMM40 gene.
 77. The kitof claim 74, wherein the kit further comprises at least one polymerase.78. The kit of claim 74, wherein the AT/GC ratio is a value in the rangefrom about 10 to about
 40. 79. The kit of claim 74, wherein the NTPscomprise dNTPs or rNTPs, and are present in a total NTP concentrationranging from about 0.5 mM to about 5 mM.
 80. The kit of claim 74,wherein dGTP is present in a concentration ranging from about 10 μM toabout 400 μM and cytidine is present in a concentration ranging fromabout 10 μM to about 400 μM.
 81. The kit of claim 74, wherein dTTP ispresent in a concentration ranging from about 700 μM to about 2000 μMand adenosine is present in a concentration ranging from about 700 μM toabout 2000 μM.
 82. The kit of claim 74 further comprising Mg²⁺, whereinthe Mg²⁺ is present in a molarity ranging from about 80% to about 150%of the molarity of total NTPs.
 83. The kit of claim 74 furthercomprising Mg²⁺, wherein the Mg²⁺ is present in a concentration rangingfrom about 1.5 mM to about 3 mM.
 84. The kit of claim 74, wherein the atleast one nucleic acid template comprises a homopolymeric segmentcomprising 10 to 40 consecutive A residues or 10 to 40 consecutive Tresidues.
 85. The kit of claim 74, wherein the at least one nucleic acidtemplate comprises a homopolymeric segment of at least 10 consecutive Tresidues.
 86. The kit of claim 74, wherein the at least one nucleic acidtemplate comprises a segment comprising (T_(n)A)_(m), (AT_(n))_(m),(TA_(n))_(m), or (A_(n)T)_(m), wherein n is 2 or greater and m is suchthat the length of the repeating A/T-rich segment is 10 or moreresidues.
 87. The kit of claim 74, wherein at least one primer comprisesa 3′-terminal sequence that specifically hybridizes to two or moreconsecutive A residues or to two or more consecutive T residues.
 88. Thekit of claim 87, wherein the at least one primer comprises a 3′-terminalsequence comprising 4 to 9 consecutive A residues or 4 to 9 consecutiveT residues.
 89. The kit of claim 74, wherein at least one primercomprises a covalently attached label.
 90. The kit of claim 77, whereinthe at least one polymerase comprises a hot-start DNA polymerase. 91.The kit of claim 74, wherein the at least one nucleic acid templatecomprises a homopolymeric segment comprising at least 12 consecutive Aresidues or at least 12 consecutive T residues.
 92. The kit of claim 74wherein the at least one nucleic acid template comprises a homopolymericsegment in a genetic locus associated with late-onset Alzheimer'sdisease.
 93. The kit of claim 74, wherein the at least one nucleic acidtemplate comprises a subset of TOMM40 sequence comprising ahomopolymeric segment of at least 10 consecutive T residues.